ES2246927T3 - BICYCLE INHIBITORS OF GLUCOGENO SINTASA QUINASA 3. - Google Patents

Abstract

New bicyclic based compounds, compositions and methods of inhibiting the activity of glycogen synthase kinase (GSK3) in vitro and of treatment of GSK3 mediated disorders in vivo are provided. The methods, compounds and compositions of the invention may be employed alone, or in combination with other pharmacologically active agents in the treatment of disorders mediated by GSK3 activity, such as in the treatment of diabetes, Alzheimer's disease and other neurodegenerative disorders, obesity, atherosclerotic cardiovascular disease, essential hypertension, polycystic ovary syndrome, syndrome X, ischemia, traumatic brain injury, bipolar disorder, immunodeficiency or cancer.

Description

Bicyclic glycogen synthase inhibitors kinase 3.

Field of the Invention

This invention relates to new compounds bicyclics that inhibit glycogen synthase kinase 3 activity (GSK3), to pharmaceutical compositions containing the compounds and to the use of the compounds and compositions, alone or in combination with other pharmaceutically active agents. The compounds and compositions provided by the present invention have utility in the treatment of activity-mediated disorders of GSK3 such as diabetes, Alzheimer's disease and others neurodegenerative disorders, obesity, cardiovascular disease atherosclerotic, essential hypertension, ovarian syndrome polycystic, syndrome X, ischemia, especially cerebral ischemia, traumatic brain injury, bipolar disorder, immunodeficiency and Cancer.

Background of the invention

Glycogen synthase kinase 3 (GSK3) is a serine / threonine kinase for which two isoforms have been identified, α and β. Woodgett, Trends Biochem Sci. , 16 : 177-81 (1991). Both isoforms of GSK3 are constitutively active in resting cells. GSK3 was originally identified as a kinase that inhibits glycogen kinase by direct phosphorylation. In the activation of insulin, GSK3 is inactivated, which allows the activation of glycogen synthase and possibly other insulin-dependent phenomena, such as glucose transport. Subsequently, it has been shown that the activity of GSK3 is also inactivated by other growth factors, such as insulin, indicated by tyrosine kinase receptors (RTK). Examples of such signaling molecules include IGF-1 and EGF. Saito et al., Biochem. J. , 303 : 27-31 (1994); Welsh et al., Biochem. J. 294 : 625-29 (1993); and Cross et al., Biochem. J. , 303 : 21-26 (1994).

Agents that inhibit the activity of GSK3 are useful in the treatment of disorders that are mediated by GSK3 activity. In addition the inhibition of GSK3 simulates the activation of growth factor signaling pathways and in consequently GSK3 inhibitors are useful in the treatment of diseases in which such routes are insufficiently active. Examples of diseases that can be described are described below. deal with GSK3 inhibitors.

Diabetes

Type 2 diabetes is a disease increasingly prevalent of aging. Is characterized initially due to lower insulin sensitivity and an elevation compensatory in circulating insulin concentrations, the latter is required to keep glucose levels in normal blood Higher insulin levels are caused by the greater secretion of pancreatic beta cells, and the resulting hyperinsulinemia is associated with complications cardiovascular diabetes. When resistance to insulin worsens, the demand in pancreatic beta cells increases stationary until the pancreas can no longer provide adequate levels of insulin, resulting levels high blood glucose Finally, the manifest hyperglycemia and hyperlipidemia, which leads to long-term devastation complications associated with diabetes, including cardiovascular diseases, kidney failure and blindness. The exact mechanism (s) that cause type 2 diabetes are (is) unknown (s), but result in poor transport of the glucose to skeletal muscle and increased glucose production liver, in addition to inadequate insulin response. The Dietary modifications are frequently ineffective, so most patients ultimately require Pharmaceutical intervention in an effort to prevent and / or slow the progression of disease complications. Many patients can be treated with one or more of the many oral antidiabetic agents available, including sulfonylureas, to increase insulin secretion. Examples of Sulfonylurea drugs include metformin for elimination of the hepatic glucose production, and troglitazone, a medication of insulin sensitization. Despite the usefulness of these agents, 30 to 40% of diabetics are not controlled by properly using these medications and require injections of insulin subcutaneously. Additionally, all of these Therapies have associated side effects. For example, the sulfonylureas can cause hypoglycemia and troglitazone may cause severe hepatoxicity Currently there is a need for new and improved drugs for the treatment of patients prediabetic and diabetic.

As described above, inhibition of GSK3 stimulates insulin-dependent processes and is consequently useful in the treatment of type 2 diabetes. Recent data obtained using lithium salts provide evidence of this perception. It has been recently described that lithium ion inhibits the activity of GSK3. Klein et al., PNAS 93 : 8455-9 (1996). Since 1924, lithium has been described as having antidiabetic effects including the ability to reduce plasma glucose levels, increase glycogen uptake, boost insulin, overregulate glucose synthase activity and stimulate glycogen synthesis in the skin. , muscle and adipocytes. However, lithium has not been widely accepted for use in inhibiting the activity of GSK3, possibly due to its documented effects on molecular targets other than GSK3. The purine analogue 5-iodotubercidine, also a GSK3 inhibitor, also stimulates glycogen synthesis and antagonizes the inactivation of glycogen synthase by glucagon and vasopressin in rat liver cells. Fluckiger-Isler et al., Biochem J 292 : 85-91 (1993); and Massillon et al., Biochem J 299 : 123-8 (1994). However, it has also been shown that this compound inhibits other serine / threonine and tyrosine kinases. Massillon et al., Biochem J 299 : 123-8 (1994).

Alzheimer disease

GSK3 is also involved in the biological pathways related to Alzheimer's disease (AD). The characteristic pathological features of AD are extracellular plaques in an abnormally processed form of the amyloid precursor protein (APP), called β-amyloid peptide (β-AP) and the development of intracellular neurofibrillar tangles containing helical paired filaments (PHF ) which are largely composed of hyperphosphorylated tau protein. GSK3 is one of a number of kinases that have been found to phosphorylate tau protein in vitro at abnormal sites characteristic of PHF tau, and is the only kinase that has been shown to do this in living cells and in animals. Lovestone et al., Current Biology 4 : 1077-86 (1994); and Brownless et al., Neuroreport 8 : 3251-3255 (1997). In addition, the GSK3 kinase inhibitor, LiCl, blocks tau hyperphosphorylation in cells. Stambolic et al., Current Biology 6 : 1664-8 (1996). Thus, the activity of GSK3 can contribute to the generation of neurofibrillar tangles and consequently to the progression of the disease. It has recently been shown that GSK3β is associated with another key protein in the pathogenesis of AD, presenilin 1 (PS1). Takashima et al., PNAS 95 : 9637-9641 (1998). Mutations in the PS1 gene lead to increased production of β-AP, but the authors also demonstrate that mutant PS1 proteins bind more strongly to GSK3β and enhance phosphorylation of tau, which is bound to the same region. of PS1.

Interestingly, it has also been shown that another GSK3 substrate, β-catenin, binds to PS1. Zhong et al., Nature 395 : 698-702 (1998). Cytosolic β-catenin is oriented to degradation after phosphorylation by GSK3 and reduced β-catenin activity is associated with increased neuronal cell sensitivity with respect to β-AP-induced neuronal apoptosis. Consequently, the greater association of GSK3? With mutant PS1 may contribute to reduced levels of? -Catenin, which have been observed in the brains of patients with mutant PS1 AD and the disease-related increase in neuronal cell death . In accordance with these observations, it has been shown that injection of antisense GSK3 but not in sense blocks the pathological effects of β-AP in neurons in vitro , resulting in a 24-hour delay at the beginning of cell death and greater cell survival of 12 to 35% in 1 hour. Takashima et al., PNAS 90 : 7789-93 (1993). In these last studies the effects on cell death are preceded (within 3 to 6 hours of the administration of β-AP) by doubling the activity of intracellular GSK3, suggesting that in addition to the genetic mechanism that increases the proximity of GSK3 to its substrate, β-AP can really increase the activity of GSK3. Other evidence of a role for GSK3 in AD is provided by the observation that the level of protein expression (but, in this case, non-specific activity) of GSK3 is increased by 50% in postsinaptosomal AD supernatants versus tissue of normal brain. Pei et al., J. Neuropathol Exp 56 : 70-78 (1997). Thus, it is believed that specific inhibitors of GSK3 will act to slow the progression of Alzheimer's disease.

Other disorders of the CNS (central nervous system)

In addition to the effects of lithium described above, there is a long history of the use of lithium to treat bipolar disorder (manic depressive syndrome). This clinical response to lithium may reflect an involvement of GSK3 activity in the etiology of bipolar disorder, in which case GSK3 inhibitors may be relevant for that indication. As corroboration of this perception, it has recently been shown that valproate, another drug commonly used in the treatment of bipolar disorder, is also a GSK3 inhibitor. Chen et al., J. Neurochemistry 72 : 1327-1330 (1999). One mechanism by which lithium and other GSK3 inhibitors can act to treat bipolar disorder is to increase the survival of neurons subjected to aberrantly high levels of excitation induced by the neurotransmitter, glutamate. Nonaka et al., PNAS 95 : 2642-2647 (1998). It is also believed that glutamate-induced neuronal excitotoxicity is a leading cause of neurodegeneration associated with acute damage, such as in cerebral ischemia, traumatic brain injury and bacterial infection. It is also believed that excessive glutamate signaling is a factor in chronic neuronal damage seen in diseases such as dementia associated with Alzheimer's, Huntington, Parkinson's, AIDS, amyotrophic lateral sclerosis (AML) and multiple sclerosis (MS). Thomas, J. Am. Geriatr. Soc. 43 : 1279-89 (1995). Consequently it is believed that GSK3 inhibitors are a useful treatment in these and other neurodegenerative diseases.

Immunological potentiation

GSK3 phosphorylates the NF-AT transcription factor and promotes its export from the nucleus, as opposed to the effect of calcineurin. Beals et al., Science 274 : 1939-33 (1997). Thus, GSK3 blocks early gene activation of immune response by NF-AT, and GSK3 inhibitors may tend to allow or prolong the activation of immune responses. Thus, it is believed that GSK3 inhibitors prolong and enhance the immunostimulatory effects of certain cytokines, and such an effect may improve the potential of those cytokines for tumor immunotherapy or even for immunotherapy in general.

Other disorders

Lithium also has other biological effects. It is a potent stimulator of hematopoiesis, both in vitro and in vivo . Hammond et al., Blood 55 : 26-28 (1980). In dogs, lithium carbonate eliminated recurrent neutropenia and normalized other blood cell parameters. Doukas et al. Exp Hematol 14 : 215-221 (1986). If these effects of lithium are mediated by GSK3 inhibition, then GSK3 specific inhibitors may have even broader therapeutic applications.

Because GSK3 inhibitors are useful in the treatment of many diseases the Identification of new GSK3 inhibitors.

Summary of the Invention

It has now been surprisingly discovered that glycogen synthase kinase 3 (GSK3) activity can be inhibited in vitro or in vivo by certain pyrimidine and pyridine based derivatives provided by the present invention. In addition to this, it has been found that the compounds of the present invention have specificity for GSK3. In accordance with the foregoing, the present invention provides new compounds, compositions and methods of inhibiting GSK3 activity in vitro and treating disorders mediated by GSK3 in vivo . In one aspect, the present invention provides new compounds that exhibit GSK3 inhibition activity of the following formula (I):

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one

in the that:

W and Z are carbon, nitrogen or sulfur optionally substituted;

X and Y are independently selected from the group consisting of nitrogen, oxygen, and carbon optionally replaced;

n is 0, 1 or 2;

A 1 and A 2 are aryl, aryloxy, arylamino or optionally substituted heteroaryl;

R1, R2, R3 and R4 are independently selected from the group consisting of hydrogen, hydroxyl and lower alkyl, lower cycloalkyl, alkylaminoalkyl, lower alkoxy, amino, alkylamino, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, aryl and heteroaryl optionally substituted;

R '1, R' 2, R '3 and R' 4 are independently selected from the group consisting of hydrogen, and optionally substituted lower alkyl;

R 5, R 6 and R 7 are selected regardless of the group consisting of hydrogen, hydroxy, halo, carboxyl, nitro, amino, amido, amidino, imido, cyano and lower alkyl substituted or unsubstituted, lower alkoxy, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroalkylcarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, alkylaminocarbonyloxy, arylaminocarbonyloxy, formyl, alkyl lower carbonyl, alkoxy lower-carbonyl, aminocarbonyl, aminoaryl, alkylsulfonyl, sulfonamido, aminoalkoxy, alkylamino, arylamino, aralkylamino, heteroarylamino, heteroaralkylamino, alkylcarbonylamino, alkylaminocarbonylamino, arylaminocarbonylamino, aralkylcarbonylamino, heteroaralkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, amidino, cycloalkyl, cycloamido, cyclothioamido, cycloamidino, heterocycloamidino, cycloimido, heterocycle, guanidinyl, aryl, biaryl, heteroaryl, heterobaryl, heterocycle, heterocycloalkyl, arylsulfonyl and arylsulfonamido;

and pharmaceutically acceptable salts of same.

The current compounds particularly Preferred and new of the invention are provided by the compounds of formula:

2

where W, X, Y, A_ {1}, A_ {2} and R 1 -R 7 have the meanings described above, and the pharmaceutically acceptable salts of the same.

The procedures, compounds and compositions of the invention can be used alone, or in combination with others pharmacologically active agents in the treatment of disorders mediated by the activity of GSK3, such as in the treatment of diabetes, Alzheimer's disease and other disorders neurodegenerative, obesity, cardiovascular disease atherosclerotic, essential hypertension, ovarian syndrome polycystic, syndrome X, ischemia, especially cerebral ischemia, traumatic brain injury, bipolar disorder, immunodeficiency or Cancer.

Detailed description of the invention

In accordance with the present invention, compounds, compositions and methods for the inhibition of glycogen synthase kinase 3 (GSK3) activity are provided, either in vitro or in vivo . In one aspect, the present invention provides novel compounds that have GSK3 inhibition activity of the following formula (I):

3

in the that:

W and Z are carbon, nitrogen or sulfur optionally substituted;

X and Y are independently selected from the group consisting of nitrogen, oxygen, and carbon optionally replaced;

n is 0, 1 or 2;

A 1 and A 2 are aryl, aryloxy, arylamino or optionally substituted heteroaryl;

R1, R2, R3 and R4 are independently selected from the group consisting of hydrogen, hydroxyl, and optionally substituted lower alkyl, cycloalkyl  lower alkylaminoalkyl, lower alkoxy, amino, alkylamino, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, aryl and heteroaryl;

R '1, R' 2, R '3 and R' 4 are independently selected from the group consisting of hydrogen, and optionally substituted lower alkyl;

R 5, R 6, R 7 and are selected regardless of the group consisting of hydrogen, hydroxy, halo, carboxyl, nitro, amino, amido, amidino, imido, cyano and lower alkyl substituted or unsubstituted, lower alkoxy, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, alkylaminocarbonyloxy, arylaminocarbonyloxy, formyl, alkyl lower carbonyl, alkoxy lower-carbonyl, aminocarbonyl, aminoaryl, alkylsulfonyl, sulfonamido, aminoalkoxy, alkylamino, arylamino, aralkylamino, heteroarylamino, heteroaralkylamino, alkylcarbonylamino, alkylaminocarbonylamino, arylaminocarbonylamino, aralkylcarbonylamino, heteroaralkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, amidino, cycloalkyl, cycloamido, cyclothioamido, cycloamidino, heterocycloamidino, cycloimido, heterocycle, guanidinyl, aryl, biaryl, heteroaryl, heterobaryl, heterocycle, heterocycloalkyl, arylsulfonyl and arylsulfonamido;

and pharmaceutically acceptable salts of same.

In a presently preferred embodiment of the invention, at least one of X and Y is nitrogen. Compounds Representative of this group include those compounds in the that one of X and Y is nitrogen and the other of X and Y is oxygen or optionally substituted carbon. Preferably, both X and Y are nitrogen.

The constituents A_ {1} and A_ {2} can be independently an aromatic ring having 3 to 10 atoms carbon in the ring and optionally 1 or more heteroatoms in the ring. Thus, in one embodiment, A_ {1} and / or A_ {2} can be optionally substituted carbocyclic aryl. Alternatively, A 1 and / or A 2 are optionally substituted heteroaryl, such such as, for example, pyridyl, pyrimidinyl, thiazolyl, indolyl, imidazolyl, oxadiazolyl, tetrazolyl, pyrazinyl, triazolyl, thiophenyl, furanyl, quinolinyl, purinyl, naphthyl, benzothiazolyl, benzopyridyl and benzimidazolyl substituted or not substituted, which can be substituted with at least one and not more than 3 replacement groups Representative substitution groups are they can select independently of the constituted group, by example, by nitro, amino, cyano, halo, thioamido, amidino, oxamidino, alkoxyamidino, imidino, guanidino, sulfonamido, carboxyl, formyl, lower alkyl, lower haloalkyl, alkoxy lower, lower haloalkoxy, lower alkoxyalkyl, alkyl lower-lower aminoalkoxy, alkyl lower carbonyl, aralkyl lower-carbonyl, heteroaralkyl lower carbonyl, alkylthio, aminoalkyl and cyanoalkyl.

In a particularly preferred embodiment current of the invention, A_ {1} and / or A_ {2} has the formula:

4

in which R_ {8} and R_ {9} are independently selected from the group consisting of hydrogen, nitro, amino, cyano, halo, thioamido, amidino, oxamidino, alkoxyamidino, imidino, guanidinyl, sulfonamido, carboxyl, formyl, lower alkyl, lower haloalkyl, lower alkoxy, lower haloalkoxy, lower alkoxyalkyl, alkyl lower-lower aminoalkoxy, alkyl lower carbonyl, aralkyl lower-carbonyl, heteroaralkyl lower carbonyl, alkylthio, aryl and aralkyl

In a particularly preferred embodiment current of the invention, A_ {1} and / or A_ {2} has the formula:

5

in which L is -NH- or -O-; Y R_ {8} and R_ {9} are independently selected from the group consisting of hydrogen, nitro, amino, cyano, halo, thioamido, amidino, oxamidino, alkoxyamidino, imidino, guanidinyl, sulfonamido, carboxyl, formyl, lower alkyl, haloalkyl lower, lower alkoxy, lower haloalkoxy, alkoxyalkyl lower, lower alkyl-lower aminoalkoxy, lower alkylcarbonyl, aralkyl lower-carbonyl, heteroaralkyl lower carbonyl, alkylthio, aryl and aralkyl. The more preferably, A 1 and / or A 2 [A] is selected from group consisting of nitropyridyl, aminonitropyridyl, cyanopyridyl, cyanothiazolyl, aminocyanopyridyl, trifluoromethylpyridyl, methoxypyridyl, methoxynitropyridyl, methoxycyanopyridyl and nitrotiazolyl

In other embodiments of the invention at least one of R 1, R 2, R 3 and R 4 may be hydrogen, or unsubstituted or substituted lower alkyl selected from the group consisting of lower haloalkyl, heterocycloaminoalkyl, and lower alkyl-lower aminoalkyl; or rent lower-lower aminoalkyl. The realizations Preferred present invention include compounds in which R 1, R 2 and R 3 are hydrogen and R 4 is selected from the group consisting of hydrogen, methyl, ethyl, aminoethyl, dimethylaminoethyl, pyridylethyl, piperidinyl, pyrrolidinylethyl, piperazinylethyl and morpholinyl ethyl.

Other current preferred compounds of the invention include compounds of formula (I) in which at least one of R 5 and R 7 is selected from the group consisting of aryl, substituted and unsubstituted heteroaryl and biaryl. In the current preferred embodiments, at least one of R5 and R 7 is a substituted or unsubstituted moiety of formula:

6

in which R_ {10}, R_ {11}, R 12, R 13 and R 14 are independently selected between the group consisting of hydrogen, nitro, amino, cyano, halo, thioamido, carboxyl, hydroxy and lower alkyl optionally substituted, lower alkoxy, lower alkoxyalkyl, haloalkyl lower, haloalkoxy lower, aminoalkyl, alkylamino, alkylthio, alkylcarbonylamino, aralkylcarbonylamino, heteroaralkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aminocarbonyl, alkyl lower-aminocarbonyl, aminoaralkyl, alkyl lower-aminoalkyl, aryl, heteroaryl, cycloheteroalkyl, aralkyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, arylcarbonyloxyalkyl, alkylcarbonyloxyalkyl, heteroarylcarbonyloxyalkyl, aralkylcarbonyloxyalkyl and heteroaralcarbonyloxyalkyl. Be obtain current particularly preferred compounds in which R 10, R 11, R 13 and R 14 are hydrogen and R 12 is select from the group consisting of halo, lower alkyl, hydroxy, lower alkoxy, lower haloalkyl, aminocarbonyl, alkylaminocarbonyl and cyano; R 11, R 13 and R 14 are hydrogen and R 10 and R 12 are independently selected from the group consisting of halo, lower alkyl, hydroxy, alkoxy lower, lower haloalkyl and cyano; R 10, R 11, R 13 and R 14 are hydrogen and R 12 is heteroaryl; R 10, R 11, R 13 and R 14 are hydrogen and R 12 is a heterocycloalkyl; and in which at least one of R_ {10}, R 11, R 12, R 13 and R 14 are halo and the remaining R 10, R 11, R 12, R 13 and R 14 are hydrogen. Preferably, at least one of R 5 and R 7 are selected of the group consisting of dichlorophenyl, difluorophenyl, trifluoromethylphenyl, chlorofluorophenyl, bromochlorophenyl, ethylphenyl, methylchlorophenyl, imidazolylphenyl, cyanophenyl, morpholinophenyl and cyanochlorophenyl.

In representative embodiments of the invention, R 6 may be substituted alkyl such as, for example, aralkyl, hydroxyalkyl, aminoalkyl, aminoaralkyl, carbonylaminoalkyl, alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl, aralkylcarbonylaminoalkyl, aminoalkoxyalkyl; substituted amino such as alkylamino, alkylcarbonylamino, alkoxycarbonylamino, arylalkylamino, arylcarbonylamino, alkylthiocarbonylamino, arylsulfonylamino, heteroarylamino, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, and heteroaralkylcarbonylamino; or substituted carbonyl such as aminocarbonyl, alkyloxycarbonyl, aryloxycarbonyl, aralkyloxycarbonyl and unsubstituted or substituted alkylaminoalkyloxycarbonyl. In other embodiments, R 6 may be selected from the group consisting of amidino, guanidino, cycloimido, heterocycloimido, cycloamido, heterocycloamido, cyclothioamido and lower heterocycloalkyl. In still other embodiments, R 6 may be aryl or heteroaryl such as, for example, pyridyl, pyrimidinyl, thiazolyl, indolyl, imidazolyl, oxadiazolyl, tetrazolyl, pyrazinyl, triazolyl, thienyl, furanyl, quinolinyl, pyrroliopyridyl, benzothiazolyl, benzopyriazyl, benzopyrolidyl substituted or unsubstituted benzotriazolyl, and benzimidazolyl. As used in this invention, representative heterocycle groups include, for example, those shown below (where the point of attachment of the substituent group, and of the other substituent groups shown below, is located at the upper left). These heterocycle groups may be additionally substituted and may be linked in various positions as will be apparent to those skilled in the art of organic and medicinal chemistry in conjunction with the description of this
invention.

7

8

9

Representative heteroaryl groups include, for example, those shown below. These heteroaryl groups may be further substituted and may be linked in various positions as will be apparent to those skilled in the art of organic and medicinal chemistry in conjunction with the description of this
invention.

10

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eleven

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12

Cycloimido and heterocycloimido groups Representative include, for example, those shown at continuation. These cycloimido and heterocycloimido can be additionally substituted and may be linked in several positions as will be apparent to specialists in techniques of organic and medicinal chemistry in conjunction with the description of this invention.

13

14

fifteen

16

Substituted amidino and heterocycloamidino groups Representative include, for example, those shown at continuation. These amidino and heterocycloamidino can be additionally replaced as will be apparent to specialists in the techniques of organic and medicinal chemistry in conjunction with the description of this invention.

17

Alkylcarbonylamino groups, alkyloxycarbonylamino, aminoalkyloxycarbonylamino and Representative substituted arylcarbonylamino include, for example, those shown below. These groups can be additionally substituted as will be apparent to specialists in the techniques of organic and medicinal chemistry in in conjunction with the description of this invention.

18

19

Representative substituted aminocarbonyl groups include, for example, those shown below. These heterocycle groups may be further substituted as it will be evident to chemistry specialists Organic and medicinal in conjunction with the description of this invention.

twenty

twenty-one

Substituted alkoxycarbonyl groups Representative include, for example, those shown at continuation. These alkoxycarbonyl groups may be substituted. additionally as will be evident to specialists in techniques of organic and medicinal chemistry in conjunction with the description of this invention.

22

2. 3

Current particularly preferred compounds of The invention includes compounds having the structure:

24

wherein W, X, Y, A 1, A 2 and R 1 -R 7 have the meanings described above, and the pharmaceutically acceptable salts thereof. Representative presently preferred compounds of this group include, for example, tert - butyl 4-chloro-2-methylthio-5,6,7,8-tetrahydropyridine [3,4-d] pyrimidin-6-carboxylate, 4- (2,4-dichlorophenyl) -2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate, 4- (2,4-dichlorophenyl) -2- (Methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] tert - butyl pyrimidin-6-carboxylate, [4- (2,4-dichlorophenyl) chlorhidate (5,6,7,8 -tetrahydropyridine [4,3-d] pyrimidin-2-yl)] {2 - [(5-nitro (2-pyridyl)) amino] ethyl} amine, {2 - [(6-amino-5-nitro hydrochloride) (2-pyridyl)) amino] ethyl} [4- (2,4-dichlorophenyl) - (5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-2-yl)] amine, 6 hydrochloride - [(2 - {[4- (2,4-dichlorophenyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-2-yl] amino} ethyl) -amino] pyridin-3- carbonitrile, 4- (2,4-dichlorophenoxy) -2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate, 4- (2,4-dichlorophenoxy ) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] -pyrimidin-6-carboxylate of tert -butyl, hydrochloride of {2 - [(6-
amino-5-nitro (2-pyridyl)) amino] ethyl} {4 - [(2,4-dichlorophenyl) amino] (5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-2-yl )}
tert -butyl amine, 4 - [(2,4-dichlorophenyl) amino] -2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-6-carboxylate, 4 - [(2 , 4-dichlorophenyl) amino] -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate, hydrochloride {2 - [(6-amino -5-nitro (2-pyridyl)) amino] ethyl} {4 - [(2,4-dichlorophenyl) -amino] (5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-2-yl )} amine, 4-[(2,4-difluorophenyl) -2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-6-carboxylic acid tert - butyl , 4 - [(2 , 4-difluorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate -
butyl, 4- (2,4-dichlorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine, 6-acetyl-4- (2,4-dichlorophenyl) - 2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] -pyrimidine, 6-acetyl-2 - ({2 - [(6-amino-5-nitro (2-pyridyl))) amino] ethyl}
amino) -4- (2,4-dichlorophenyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine, and 6 - [(2 - {[6-acetyl-4- (2,4 -dichlorophenyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-2-yl] amino} ethyl) -amino] pyridin-3-carbonitrile.

In another aspect, the invention provides compositions comprising an amount of a compound of formula (I) effective to modulate the activity of GSK3 in a human subject or animal when administered to it, together with a vehicle pharmaceutically acceptable.

In still other embodiments, the invention provides procedures for inhibition of GSK3 in a subject human or animal, which comprises administration to the human subject or animal of an inhibitory amount of GSK3 of a compound of structure (I).

The present invention further provides treatment procedures of human or animal subjects that suffer from disorders mediated by GSK3 in a human subject or animal, which comprises administration to a human or animal subject of a therapeutically effective amount of a compound of formula (I) above, either alone or in combination with other agents therapeutically active.

In still other embodiments the present invention provides compounds of formulas I, IV and V, as described above, for use as a pharmaceutical compound, as well as also procedures for using these compounds in manufacturing of a medication for the treatment of diabetes disease Alzheimer's and other neurodegenerative disorders, obesity, atherosclerotic cardiovascular disease, essential hypertension, polycystic ovary syndrome, syndrome X, ischemia, especially cerebral ischemia, traumatic brain injury, bipolar disorder, immunodeficiency or cancer

As used above and in any other part of this invention, the following terms present the meanings defined below:

"Glycogen synthase kinase 3" and "GSK3" are used interchangeably in this invention to refer to any protein that has more than 60% amino acid sequence homology between positions 56 and 340 of the beta amino acid sequence of Human GSK3 (Genbank Accession number L33801). To determine the percentage homology of two amino acid or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, hiatus can be introduced into the sequence of a polypeptide or nucleic acid for optimal alignment with the other polypeptide or nucleic acid). The amino acid or nucleotide residues in the corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the other sequence, then the molecules are homologous in that position (ie, as "homology" of amino acid or acid is used in this invention nucleic acid is equivalent to "identity" of amino acid or nucleic acid). The percentage homology between the two sequences is a function of the number of identical positions divided by the sequences (ie,% of homology = number of identical positions / total number of positions x 100). GSK3 was initially identified by phosphorylation of glycogen synthase as described by Woodgett et al., Trends Biochem. Sci. , 16 : 177-81 (1991), incorporated herein by reference. By modulating the activity of the GSK3 kinase, the activities downstream of the activity of GSK3 can be inhibited, or alternatively. For example, when GSK3 activity is inhibited, glycogen synthase can be activated, resulting in increased glycogen production. It is also known that GSK3 acts as a kinase in a variety of other contexts including, for example, phosphorylation of c-jun, β-catenin and tau protein. It is understood that inhibition of GSK3 kinase activity can lead to a variety of effects in a variety of biological contexts. The invention, however, is not limited by any mechanism theory on how the
invention.

"GSK3 inhibitor" as used in this invention refers to a compound that shows an IC50 with respect to GSK3 of no more than about 100 µM and more typically no more than about 50 µM, measured in the assay no GSK3 inhibitory activity cells generally described later. "IC_ {50}" is that concentration of inhibitor that reduces the activity of an enzyme (for example, GSK3) to Half the maximum level. It has been discovered that compounds Representative of the present invention show activity inhibitor against GSK3. The compounds of the present invention preferably show an IC 50 with respect to GSK3 of no more than about 10 µM, more preferably, no more than about 5 µM, even more preferably no more than about 1 µM, and most preferably, no more than approximately 200 nM, measured in the GSK3 kinase assay without cells.

"Optionally substituted" refers to the Hydrogen replacement with a monovalent or divalent radical. Suitable substitution groups include, for example, hydroxyl, nitro, amino, imino, cyano, halo, uncle, thioamido, amidino, imidino, oxo, oxamidino, methoxamidino, imidino, guanidino, sulfonamido, carboxyl, formyl, lower alkyl, lower haloalkyl, alkoxy lower, lower haloalkoxy, lower alkoxyalkyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, alkylthio, aminoalkyl, cyanoalkyl and the like.

The substitution group can be itself replaced. The substituted group in the substitution group can be carboxyl, halo; nitro, amino, cyano, hydroxyl, alkyl lower, lower alkoxy, aminocarbonyl, -SR, thioamido, -SO 3 H, -SO 2 R or cycloalkyl, where R is typically hydrogen, hydroxyl or lower alkyl.

When the substituted substituent includes a linear chain group, substitution can take place well within the chain (for example, 2-hydroxypropyl, 2-aminobutyl, and the like) or at the end of the chain (for example, 2-hydroxyethyl, 3-cyanopropyl, and the like). Substituents substituted may be straight chain, branched or cyclic carbon atoms or heteroatoms bonded together covalent

"Lower alkyl" as used in this invention refers to branched or straight chain alkyl groups comprising one to ten carbon atoms that are unsubstituted or substituted, for example, with one or more halogens, hydroxyls or other groups. , including, for example, methyl, ethyl, propyl, isopropyl, n- butyl, t- butyl, neopentyl, trifluoromethyl, pentafluoroethyl and the like.

"Alkylene" refers to a radical saturated linear chain or branched chain aliphatic divalent that has 1 to 20 carbon atoms. Alkylenyl groups Typical used in compounds of the present invention are groups lower alkylenyl having 1 to about 6 atoms of carbon in your skeleton. "Alkenyl" refers in this invention to linear, branched or cyclic chain radicals that They have one or more double bonds and 2 to 20 carbon atoms. "Alkynyl" refers herein to chain radicals. linear, branched or cyclic that has one or more triple bonds and  from 2 to 20 carbon atoms.

"Lower alkoxy" as used in this invention refers to RO- in which R is lower alkyl. Representative examples of lower alkoxy groups include methoxy, ethoxy, t- butoxy, trifluoromethoxy and the like.

"Cycloalkyl" refers to an alkyl, heterocyclic or carbocyclic, mono- or polycyclic substituent. Typical cycloalkyl substituents have 3 to 8 atoms of the skeleton (i.e., of the ring) in which each atom of the skeleton is carbon or a heteroatom. The term "heterocycloalkyl" refers in this invention to cycloalkyl substituents having 1 to 5, and more typically, 1 to 4 heteroatoms in the ring structure. Suitable heteroatoms used in compounds of the present invention are nitrogen, oxygen and sulfur. Representative heterocycloalkyl moieties include, for example, morpholino, piperazinyl, piperadinyl and the like. Carbocycloalkyl groups are cycloalkyl groups in which all ring atoms are carbon. When used together with cycloalkyl substituents, the term "polycyclic" refers in this invention to condensed and non-cyclic alkyl cyclic structures.
condensed

"Halo" refers in this invention to a halogen radical, such as fluorine, chlorine, bromine or iodine. "Haloalkyl" refers to an alkyl radical substituted with one or more halogen atoms. The term "haloalkyl lower "refers to a substituted lower alkyl radical with one or more halogen atoms. The term "haloalkoxy" is refers to an alkoxy radical substituted with one or more atoms of halogen The term "lower haloalkoxy" refers to a lower alkoxy radical substituted with one or more atoms of halogen

"Aryl" refers to monocyclic and polycyclic aromatic groups having 3 to 14 carbons or heteroatoms in the skeleton, and includes both carbocyclic and heterocyclic aryl groups. Aryl groups are carbocyclic aryl groups in which all ring atoms in the aromatic ring are carbon. The term "heteroaryl" refers in this invention to aryl groups having 1 to 4 heteroatoms as ring atoms in an aromatic ring, the rest of the ring atoms being carbon atoms. When used together with aryl substituents, the term "polycyclic" refers in this invention to condensed and uncondensed cyclic structures in which at least one cyclic structure is aromatic, such as, for example, benzodioxozole (which has a condensed heterocyclic structure with a phenyl group, that is 100 , naphthyl, and the like. Example aryl moieties used as substituents in compounds of the present invention include phenyl, pyridyl, pyrimidinyl, thiazolyl, indolyl, imidazolyl, oxadiazolyl, tetrazolyl, pyrazinyl, triazolyl, thiophenyl, furanyl, quinolinyl, purinyl, naphthyl, benzothiazolyl and benzopyridyl and the like benzopyridyl .

"Aralkyl" refers to an alkyl group substituted with an aryl group. Typically, aralkyl groups used in the compounds of the present invention have 1 to 6 carbon atoms incorporated into the alkyl part of the group aralkyl Suitable aralkyl groups used in compounds of the present invention include, for example, benzyl, picolyl and Similar.

"Amino" refers in this invention to group -NH_ {2}. The term "alkylamino" refers in this invention to the group -NRR ', where R and R' are each selected independently between hydrogen or a lower alkyl. The term "arylamino" refers in this invention to the group -NRR 'where R is aryl and R 'is hydrogen, a lower alkyl, or an aryl. He term "aralkylamino" refers in this invention to the group -NRR 'where R is a lower aralkyl and R' is hydrogen, an alkyl  lower, an aryl or a lower aralkyl.

The term "arylcycloalkylamino" refers to in this invention to the group aryl-cycloalkyl-NH-, where cycloalkyl is a divalent cycloalkyl group. Typically, cycloalkyl has 3 to 6 skeleton atoms, of which Optional 1 to about 4 are heteroatoms. The term "aminoalkyl" refers to an alkyl group that is terminally substituted with an amino group.

The term "alkoxyalkyl" refers to the group -alq_ {1} -O-alq_ {2}, where Alk 1 is alkylene or alkenyl, and Alk 2 is alkyl or alkenyl The term "lower alkoxyalkyl" refers to a alkoxyalkyl where alq1 is lower alkylene or alkenyl lower, and alq2 is lower alkyl or lower alkenyl. HE term "aryloxyalkyl" refers to the group -alkylene-O-aryl. The term "aralkoxyalkyl" refers to the group -alkylene-O-aralkyl, where Aralkyl is a lower aralkyl.

The term "alkoxyalkylamino" refers to this invention to the group -NR- (alkoxyalkyl), where R is typically hydrogen, lower aralkyl or lower alkyl. The term "lower aminoalkoxyalkyl" refers in this invention to a  aminoalkoxyalkyl in which the alkoxyalkyl is an alkoxyalkyl lower.

The term "aminocarbonyl" refers to this invention to the group -C (O) -NH2. "Substituted aminocarbonyl" refers in this invention to group -C (O) NRR 'where R is lower alkyl and R' is hydrogen or a lower alkyl. The term "arylaminocarbonyl" refers in this invention to the group -C (O) -NRR 'where R is an aryl and R' is hydrogen, lower alkyl or aryl. "Aralkylaminocarbonyl" refers in this invention to the group -C (O) -NRR 'where R is lower aralkyl and R' it is hydrogen, lower alkyl, aryl or lower aralkyl.

"Aminosulfonyl" refers in this invention to the group -S (O) 2 -NH 2. "Substituted aminosulfonyl" refers in this invention to group -S (O) 2 -NRR 'where R is lower alkyl and R 'is hydrogen or a lower alkyl. The term "aralkylaminosulfonylaryl" refers in this invention to group -aryl-S (O) 2 -NH-aralkyl, where the aralkyl is lower aralkyl.

"Carbonyl" refers to the divalent group -CO)-.

"Carbonyloxy" generally refers to group -C (O) -O-. Such groups include esters, -C (O) -O-R, where R it is lower alkyl, cycloalkyl, aryl or lower aralkyl. He term "carbonyloxycycloalkyl" is generally referred to in this invention to a "carbonyloxycarbocycloalkyl" and to a "carbonyloxyheterocycloalkyl", that is, where R is a carbocycloalkyl or heterocycloalkyl, respectively. The term "arylcarbonyloxy" refers in this invention to the group -C (O) -O-aryl, where aryl is a mono- or polycyclic carbocycloaryl or heterocycloaryl. He The term "aralkylcarbonyloxy" refers in this invention to -C (O) -O-aralkyl group, where the aralkyl is lower aralkyl.

The term "sulfonyl" refers in this invention to the group -SO2 -. "Alkylsulfonyl" refers to a substituted sulfonyl of the structure -SO2R- in which R is I rent. Alkylsulfonyl groups used in compounds of the present invention are typically alkyl groups lower sulfonyl having 1 to 6 atoms of carbon in its skeleton structure. So, groups Typical alkylsulfonyl used in compounds of the present invention include, for example, methylsulfonyl (ie, where R is methyl), ethylsulfonyl (i.e., where R is ethyl), propylsulfonyl (ie, where R is propyl) and the like. He term "arylsulfonyl" refers in this invention to the group -SO2 -aryl. The term "aralkylsulfonyl" refers in this invention to the group -SO2 -aralkyl, in which the aralkyl is lower aralkyl. The term "sulfonamido" refers in this  invention to -SO2 NH2.

As used in this invention, the term "carbonylamino" refers to the divalent group -NH-C (O) - in which the hydrogen atom of the Amide nitrogen from the carbonylamino group can be replaced by a lower alkyl, aryl or lower aralkyl group. Such groups include residues such as carbamate esters (-NH-C (O) -O-R) and amides -NH-C (O) -NR'-R, where R and R 'are straight or branched chain lower alkyl, cycloalkyl, or aryl or lower aralkyl. The term "alkyl lower-carbonylamino "refers to alkylcarbonylamino where R is a lower alkyl having from 1 to approximately 6 carbon atoms in their skeleton structure. The term "arylcarbonylamino" refers to the group -NH-C (O) -R where R is a aryl Similarly, the term "aralkylcarbonylamino" is refers to carbonylamino where R is a lower aralkyl.

As used in this invention, the term "guanidino" or "guanidilo" refers to derived residues of guanidine, H 2 N-C (= NH) -NH 2. Such remains include those attached to the nitrogen atom that carries the formal double bond (position "2" of guanidine, for example, diaminomethylene amine, (H 2 N) 2 C = NH-) and those attached to any of the nitrogen atoms they carry a formal simple link (positions "1" and / or "3" of the guanidine for example H 2 N-C (= NH) -NH-). Atoms of hydrogen in any of the nitrogen can be replaced by a suitable substituent, such as lower alkyl, aryl, or lower aralkyl.

As used in this invention, the term "amidino" refers to the remains R-C (= N) -NR'- (the radical being in the nitrogen "N1") and R (NR ') C = N- (the nitrogen radical "N2"), where R and R 'can be hydrogen, lower alkyl, aryl or lower aralkyl).

The compounds of the present invention can be easily synthesize using the procedures described in this invention, or other procedures, which are well known in the technique.

The GSK3 inhibitor compounds of the present invention can be purified using known procedures, such as, for example, chromatography, crystallization and Similar.

The compounds of the present invention preferably show inhibitory activity that is relatively substantially selective with respect to GSK3, as compared to at least one other type of kinase. As used in this invention, the term "selective" refers to a relatively greater potency for inhibition against GSK3, compared to at least one other type of kinase. Preferably, the GSK3 inhibitors of the present invention are selective with respect to GSK3, compared to at least two other types of kinases. Kinase activity assays for kinases other than GSK3 are generally known. See, for example, Havlicek et al., J. Med. Chem. , 40 : 408-12 (1997), incorporated herein by reference. GSK3 selectivity can be quantified according to the following: GSK3 selectivity = IC_ {50 \ (other \ kinase)} / CI_ {50 \ (GSK3)}, where a GSK3 inhibitor is selective for GSK3 when IC_ {50 \ (other \ kinase)}> IC_ {50 \ (GSK3)}. Thus, an inhibitor that is selective for GSK3 shows a selectivity for GSK3 greater than 1 time with respect to the inhibition of a kinase other than GSK3. As used in this invention, the term "other kinase" refers to a kinase other than GSK3. Such selectivities are generally measured in the cell-free assay described in example 20.

Typically, GSK3 inhibitors of the present invention show a selectivity of at least about 2 times (i.e., IC_ {50 \ (other \ kinase)} / IC_ {50 \ (GSK3)}) for GSK3, compared to another kinase and more typically show a selectivity of at least about 5 times. Normally, GSK3 inhibitors of the present invention show a selectivity for GSK3, compared to at less another kinase, at least about 10 times, so desirable at least about 100 times and more preferably, at least about 1000 times.

The inhibitory activity of GSK3 can be detected easily using the assays described in this invention, as well as also trials known in general by specialists in the technique. Sample procedures for the identification of GSK3 specific inhibitors include both kinase assays GSK3 without cells as cell-based. A GSK3 Kinase Assay no cells detect inhibitors that act by interaction direct with the GSK3 polypeptide while a kinase assay GSK3 based cells can identify inhibitors that work either by direct interaction with GSK3 itself, or by interference with GSK3 expression or with processing post-translational required to produce GSK3 active mature.

In general, a GSK3 kinase assay without cells It can be easily carried out by: (1) incubation of GSK3 with a peptide substrate, radioactively labeled ATP (such as, by example, γ33 P- or γ32 P-ATP, both available from Amersham, Arlington Heights, Illinois), magnesium ions and optionally one or more candidate inhibitors; (2) incubation of the mixture during a period of time to allow phosphate incorporation radioactively labeled on the peptide substrate by activity of GSK3; (3) transfer of all or part of the mixture of enzymatic reaction to a separate vessel, typically a microtiter well containing a uniform amount of a capture ligand that is capable of binding an anchor ligand on the peptide substrate; (4) wash to remove marked ATP radioactively unreacted; then (5) quantification of the amount of 33 P or 32 P remaining in each well. This amount represents the amount of radioactively labeled phosphate in the peptide substrate. Inhibition is observed as a reduction in the incorporation of radioactively labeled phosphate into the substrate peptide

Peptide substrates suitable for use in the Cellless assay can be any peptide derivative, synthetic polypeptide or peptide that can be phosphorylated by GSK3 in the presence of an appropriate amount of ATP. Peptide Substrates suitable may be based on portions of the sequences of several natural protein substrates of GSK3, and they can also contain N-terminal modifications or extensions or  C-terminals including spacer sequences and anchor ligands. Thus, the peptide substrate can reside within a longer polypeptide, or it can be an isolated peptide designed for phosphorylation by GSK3.

For example, a peptide substrate may be designed based on a sub-sequence of the CREB DNA binding protein, such as the SGSG-linked CREB peptide sequence within the CREB DNA binding protein described by Wang et al., Anal. Biochem , 220 : 397-402 (1994), incorporated herein by reference. In the assay described by Wang et al., The C-terminal serine on the SXXXS motif of the CREB peptide is enzymatically prefosphorylated by cAMP-dependent protein kinase (PKA), a step that is required to make the N-terminal serine on the phosphorylated motif by GSK3. Alternatively, a modified CREB peptide substrate can be used that has the same SXXXS motif and also contains an N-terminal anchor ligand, but that is synthesized with its pre-phosphorylated C-terminal serine (such substrate is commercially available by part of Chiron Technologies PTY Ltd., Clayton, Australia). Phosphorylation of the second serine in the SXXXS motif during peptide synthesis eliminates the need to enzymatically phosphorylate that residue with PKA as a separate step, and the incorporation of an anchor ligand facilitates the capture of the peptide substrate after its reaction with GSK3 .

Usually a peptide substrate used for a kinase activity assay may contain one or more sites that are phosphorylable by GSK3, and one or more other sites that are phosphorylable by other kinases, but not by GSK3. So these other sites can be prephosphorylated in order to create a motive which is phosphorylable by GSK3. The term "prephosphorylated" is refers in this invention to phosphorylation of a substrate peptide with phosphate not radiolabelled before carrying out a Kinase assay used that substrate peptide. Such phosphorylation is can be carried out conveniently during the synthesis of peptide substrate.

The CREB peptide bound to SGSG may be linked to an anchor ligand, such as biotin, where the serine is prefosphorylate near the term C between P and Y. As used in this invention, the term "anchor ligand" refers to a ligand that may be attached to a peptide substrate to facilitate the capture of the peptide substrate in a capture ligand, and that acts to keep the peptide substrate in place during washing stages, also allowing the removal of the marked ATP Radioactively unreacted. An example anchor ligand is the biotin The term "capture ligand" refers to this invention to a molecule that can bind an anchor ligand with high affinity, and that is attached to a solid structure. Examples of bound capture ligands include, for example, wells of microtitles coated with avidin or streptavidin or spheres of agarose Capture ligatures bearing spheres can be additionally combine with a scintillation liquid to provide a means for the detection of the labeled substrate peptide radioactively captured, or scintillation fluid can be added to the peptide captured in a last stage.

The radioactively labeled peptide substrate is can quantify in a scintillation counter using procedures known. The signal detected in the scintillation counter will be proportional to the activity of GSK3 if the enzymatic reaction has took place in conditions where only a limited part (for example, less than 20%) of the peptide substrate is phosphorylated. If a inhibitor is present during the reaction, the activity of GSK3 will be reduced, and a smaller amount of phosphate labeled radioactively it will therefore be incorporated into the peptide substrate. Hence a lower scintillation signal will be detected. In consequently, the inhibitory activity of GSK3 will be detected as a reduction in the scintillation signal, compared to the observed in a negative control, where no inhibitor is present during the reaction This essay is described in more detail in the example 265 below.

A GSK3 kinase activity assay based in cells typically uses a cell that can express both GSK3 as a substrate of GSK3, such as, for example, a cell transformed with genes encoding GSK3 and its substrate, including regulatory control sequences for gene expression. In performing the cell-based assay, the cell capable of expressing the genes is incubated in the presence of a compound of the present invention The cell is lysed, and the proportion is determined substrate in phosphorylated form, for example, by observation of its mobility with respect to the unphosphorylated form in SDS PAGE or by determining the amount of substrate that is recognized by an antibody specific for the phosphorylated form of the substrate. The amount of phosphorylation of the substrate is an indication of the inhibitory activity of the compound, that is, the inhibition as a decrease in phosphorylation compared with the test carried out without inhibitor present. Activity GSK3 inhibitor detected in a cell-based assay can be due, for example, to the inhibition of GSK3 expression or by inhibition of the kinase activity of GSK3.

Thus, cell-based assays can also be used to specifically analyze the activities that are involved in the inhibition of GSK3, such as, for example, inhibition of tau protein phosphorylation, potentiation of insulin signaling, and the like. For example, to check the ability of a GSK3 inhibitor to inhibit Alzheimer's phosphorylation of microtubule-associated tau protein, cells can be co-transfected with human GSK3β and human tau protein, then incubated with one or more candidate inhibitors. Different cell lines and mammalian expression vectors can be used for this type of assay. For example, both COS cells can be transfected with a human GSK3? Expression plasmid according to the protocol described by Stambolic et al ., 1996, Current Biology 6 : 1664-68, which is incorporated herein by reference, as an expression plasmid such as pSG5 containing human tau protein coding sequence in an early SV40 promoter. See also Goedert et al., EMBO J. , 8 : 393-399 (1989), which is incorporated herein by reference. Alzheimer's phosphorylation of tau can be readily detected with a specific antibody such as, for example, AT8, which is available from Polymedco Inc. (Cortlandt Manor, New York) after cell lysing. This essay is described in more detail in the examples, below.

Similarly, the ability of GSK3 inhibitor compounds to enhance insulin signaling by glycogen synthase activation can be readily ascertained using a cell-based glycogen synthase activity assay. This assay uses cells that respond to insulin stimulation by increasing glycogen synthase activity, such as the CHO-HIRC cell line, which overexpresses the wild-type insulin receptor (approx. 100,000 binding sites / cell). The CHO-HIRC cell line can be generated as described by Moller et al., J. Biol.Chem. , 265: 14979-14985 (1990) and Moller et al., Mol. Endocrinol. , 4: 1183-1191 (1990), both are incorporated herein by reference. The assay can be carried out by incubating CHO-HIRC cells with serum deficit in the presence of different concentrations of compounds of the present invention in the medium, followed by cell lysis at the end of the incubation period. Glycogen synthase activity can be detected in the lysate as described by Thomas et al., Anal. Biochem , 25: 486-499 (1968). The glycogen synthase activity is computed for each sample as a percentage of the maximum glycogen synthase activity, as described by Thomas et al., See above , and is represented as a function of the concentration of candidate GSK3 inhibitor. The concentration of candidate GSK3 inhibitor that increased glycogen synthase activity to half its maximum level (i.e., EC50) can be calculated by adjusting a four-parameter sigmoidal curve using routine curve fitting procedures They are well known to those skilled in the art. This is described in more detail in example 266, below.

GSK3 inhibitors can be easily classified for in vivo activity such as, for example, using procedures that are well known to those skilled in the art. For example, candidate compounds that have potential therapeutic activity in the treatment of type 2 diabetes can be easily identified by detecting a capacity to improve glucose tolerance in animal models of type 2 diabetes. Specifically, one can Dosing the candidate compound using any of several routes prior to administration of a diabetic mouse glucose bolus (e.g., KK, db / db, ob / ob) or diabetic rats (e.g., Zucker Fa / Fa or GK). After administration of the candidate compound and glucose, blood samples are taken at preselected time intervals and evaluated for serum glucose and insulin levels. Increased administration of glucose can be considered in the absence of elevated levels of endogenous insulin secretion as insulin sensitization and may be indicative of the efficacy of the compound. A detailed description of this test is given below in the examples.

The compounds of the present invention can be use in the form of salts derived from inorganic acids or organic These salts include, but are not limited to, the following: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, canforate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecyl sulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, iohydrate, 2-hydroxyenesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, sulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, p-toluenesulfonate and undecanoate. Also, you can quaternize the basic nitrogen containing groups with agents such as lower alkyl halides, such as chlorides, bromides and methyl, ethyl, propyl and butyl iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates, halides of long chain such as chlorides, bromides and decyl iodides, lauryl, myristyl and stearyl, aralkyl halides such as bromides of benzyl and phenethyl, and others. With this you get products soluble or dispersible in water or oil.

Examples of acids that can be used to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid and organic acids such as oxalic acid, maleic acid, succinic acid and citric acid. Basic addition salts can be prepared in situ during the final isolation and purification of the compounds of formula (I), or separately by reacting carboxylic acid residues with a suitable base such as hydroxide, carbonate or bicarbonate of a metal cation pharmaceutically acceptable or with ammonia, or a primary, secondary or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, aluminum and the like, as well as non-toxic ammonium, quaternary ammonium and amine cations. , including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and the like. Other representative organic amines useful for the formation of base addition salts include diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

The compounds of the present invention can be manage by a variety of routes that include routes from enteric, parenteral and topical administration. For example, modes Suitable administration include orally, subcutaneously, transdermal, transmucosal, iontophoretic, intravenous, intramuscular, intraperitoneal, intranasal, subdural, rectal and Similar.

In accordance with other embodiments of the present invention, a composition comprising compound is provided GSK3 inhibitor of the present invention, together with a vehicle or pharmaceutically acceptable excipient.

Pharmaceutically acceptable excipients suitable include processing agents and modifiers and drug release enhancers, such as, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-? -cyclodextrin, polyvinylpyrrolidinone, low melting waxes, resins ion exchange, and the like, as well as combinations of  any of two or more thereof. Other described pharmaceutically acceptable excipients at "Remington's Pharmaceutical Sciences ", Mack Pub. Co., New Jersey (1991), incorporated into this invention as a reference.

Pharmaceutical compositions containing GSK3 inhibitor compounds of the present invention can be in any form suitable for the intended procedure of administration, including, for example, a solution, a suspension, or an emulsion. Liquid vehicles are typically used in the preparation of solutions, suspensions and emulsions. Liquid vehicles contemplated for use in the practice of present invention include, for example, water, saline, Pharmaceutical organic solvent (s) Acceptable (s), pharmaceutically acceptable oils or fats and  similar, as well as mixtures of two or more thereof. He Liquid vehicle may contain other pharmaceutically additives suitable acceptable such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, agents thickeners, viscosity regulators, stabilizers and Similar. Suitable organic solvents include, for example, monohydric alcohols, such as ethanol and polyhydric alcohols, such as glucoles. Suitable oils include, for example, oil soy, coconut oil, olive oil, safflower oil, oil of cottonseed, and the like. For administration via parenterally, the vehicle can also be an oily ester such as ethyl oleate, isopropyl myristate and the like. The compositions of the present invention may also be in the form of microparticles, microcapsules, microsomal encapsulates and the like, as well as combinations of any two or More of them.

The compounds of the present invention can be administer orally, parenterally, sublingually, by aerosol for inhalation, rectal or topical in unit formulations of Dosage containing vehicles, adjuvants and potadores conventional non-toxic pharmaceutically acceptable as wish. Topical administration may also involve the use of transdermal administration such as patches transdermal or iontophoresis devices. The parenteral term as used in this invention includes injections via subcutaneous, intravenous injection, intramuscularly, intrasternal, or infusion techniques.

Injectable preparations can be formulated, by for example, sterile injectable aqueous or oleaginous suspensions according to the known technique using dispersing agents or suitable humectants and suspending agents. The preparation Sterile injectable can also be a solution or suspension sterile injectable in a diluent or solvent parenterally acceptable non-toxic, for example, as a solution in 1,3-propanediol. Between carriers and solvents Acceptable that can be used are water, Ringer's solution, and isotonic sodium chloride solution. They are also used conventionally sterile fixed oils as a solvent or medium of suspension Any fixed oil can be used for this purpose. mild including synthetic mono- or diglycerides. In addition, they are from I use fatty acids, such as oleic acid, in the preparation of injectable

Suppositories can be prepared for rectal administration of the drug by mixing the drug with a suitable non-irritating excipient such as butter cocoa and polyethylene glycols that are solid at ordinary temperatures but liquids at the temperature of the rectum and therefore will melt in the rectum and will release the drug.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound can be administered with at least one inert diluent such as sucrose lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, for example, lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills with coatings can be additionally prepared
Enteric

Liquid dosage forms for oral administration may include emulsions, solutions, pharmaceutically acceptable suspensions, syrups and elixirs that they contain inert diluents commonly used in the art, such like water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifiers and agents suspension, cyclodextrins, and sweeteners, flavorings and perfuming agents

In accordance with other embodiments, this invention provides methods for activity inhibition of GSK3 in a human or animal subject, said method comprises administration to a subject of an amount of a compound GSK3 inhibitor having structure (I), (IV) or (V) (or composition comprising such a compound) effective to inhibit the GSK3 activity in the subject. Other embodiments provided procedures for the treatment of a cell or disorder mediated by GSK3 in a human or animal subject, which comprises the administration to the cell or to the human or animal subject of a amount of an effective compound or composition of the invention to inhibit the activity of GSK3 in the cell or subject. Preferably, the subject will be a human or non-animal animal subject. human. Inhibition of GSK3 activity includes elimination detectable from the activity of GSK3 well compared to a control or compared to the activity of GSK3 expected

Effective amounts of the compounds of the invention usually include any sufficient quantity to detectably inhibit the activity of GSK3 by any of the assays described in this invention, by other GSK3 kinase activity assays known to specialists in the art or by detecting a relief of symptoms in a subject afflicted with a disorder mediated by GSK3

GSK3-mediated disorders that can be treat according to the invention include any disorder biological or medical in which the activity of GSK3 is involved or in which the inhibition of GSK3 potentiates signaling by a route that is characteristically defective in the disease that It is going to be treated. The condition or disorder may be caused or characterized by abnormal GSK3 activity. Disorders mediated by Representative GSK3 include, for example, type 2 diabetes, Alzheimer's disease and other neurodegenerative disorders, obesity, atherosclerotic cardiovascular disease, hypertension essential, polycystic ovary syndrome, syndrome X, ischemia, especially cerebral ischemia, traumatic brain injury, Bipolar disorder, immunodeficiency, cancer and the like.

The successful treatment of a subject of agreement with the invention can lead to the induction of a reduction or symptom relief in a subject afflicted with a disorder medical or biological, for example, to stop the progression Additional disorder, or disease prevention. So Well, for example, the treatment of diabetes can lead to a reduction in glucose or HbA1c levels in the patient. Similarly, the treatment of Alzheimer's disease can give result in a reduction in the speed of the progression of the disease, detected, for example, by measurement of a reduction in the rate of increase in dementia.

The amount of active substance that can be combine with vehicle materials to produce a form of Single dosage will vary depending on the treated host and the specific mode of administration. It will be understood, however, that the specific dose amount for any given patient depend on a variety of factors including the activity of the specific compound used, age, body weight, general health, sex, diet, time of administration, route of administration, excretion rate, drug combination, and severity of certain disease that undergoes therapy. The amount therapeutically effective for a given situation you can easily determine by routine experimentation and is within the experience and judgment of the ordinary practitioner.

For the purposes of the present invention, a Therapeutically effective dose will usually be about 0.1 mg / kg / day to about 100 mg / kg / day, preferably from about 1 mg / kg / day to about 20 mg / kg / day, and most preferably about 2 mg / kg / day at about 10 mg / kg / day of a GSK3 inhibitor compound of the present invention, which can be administered in one or multiple dose.

The compounds of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other liquid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any physiologically acceptable and metabolizable, non-toxic lipid capable of forming liposomes can be used. The compositions present in the form of liposome may contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients and the like. Preferred lipids are phospholipids and phosphatidylcholines (lecithins), both natural and synthetic. Methods for forming liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology , Volume XIV, Academic Press, New York, NW, page 33 and following (1976).

Although the compounds of the invention can be administer as the active pharmaceutical agent alone, these they can also be used in combination with other and other agents used in the treatment of disorders. Representative Agents useful in combination with the compounds of the invention for Type 2 diabetes treatment include, for example, insulin, troglitazone, rosiglitazone, pioglitazone, glipicide, metformin, acarbose and the like. Representative agents useful in combination with the compounds of the invention for treatment of Alzheimer's disease include, for example, donepezil, Tacrine and the like. Representative agents useful in combination with the compounds of the invention for the treatment of disorder Bipolar include, for example, lithium salts, valproate, Carbamazepine and the like. A representative agent useful in combination with the compounds of the invention for treatment of stroke is, for example, plasminogen activator in tissue.

When additional active agents are used in in combination with the compounds of the present invention, the Additional active agents can usually be used in therapeutic amounts as indicated in PHYSICIANS 'DESK REFERENCE (PDR) 53rd edition (1999), which is incorporated into this invention as a reference, or such therapeutically amounts useful as would be known by a person skilled in the art.

The compounds of the invention and the others therapeutically active agents can be administered in the maximum recommended clinical dosage or at lower doses. The Dosage amounts of the active compounds in the Compositions of the invention can be varied so that obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient. The combination can be administered as compositions by separately or as a single dosage form that contains both Agents When administered as a combination, agents Therapeutics can be formulated as separate compositions that are administered at the same time or different times, or agents Therapeutics can be administered as a unique composition.

The above and other aspects of the invention can be better understood in relation to the following examples representative.

Examples Example 1 Characterization and purification procedures

The compounds of the present invention were characterized by high performance liquid chromatography (HPLC) using a Waters Millenium chromatography system with a 2690 separation module (Mildford, Massachusetts). The analytical columns were reverse phase Alltima C-18, 4.6 x 250 mm from Alltech (Deerfield, Illinois). An elution gradient was used, typically starting with 5% acetonitrile / 95% water and progressing to 100% acetonitrile over a period of 40 minutes. All solvents contained 0.1% trifluoroacetic acid (TFA). Compounds were detected by absorption with ultraviolet (UV) light at 220 or 254 nm. The solvents for HPLC were from Burdick and Jackson (Muskegan, Michigan) or from Fisher Scientific (Pittsburg, Pennsylvania). In some cases, purity was analyzed by thin layer chromatography (TLC) using glass or plastic coated silica gel plates, such as, for example, Baker-Flex Silica Gel 1B2-F flexible sheets. TLC results are detected
They took visually easily in ultraviolet light, or using iodine vapor and other well-known tinting techniques.

Spectrometry analysis of masses in a Fisons electrospray mass spectrometer VG All the masses that occur are those of ions of origin protonated

Resonance analysis was carried out nuclear magnetic (NMR) with a 300 MHz NMR equipment Varian (Stick Alto, California). The spectral reference was TMS or the known chemical displacement of the solvent. They experienced some samples of compound at elevated temperatures (i.e. 75 ° C) to promote greater sample solubility).

The purity of some of the compounds in the invention was analyzed by elementary analysis (Desert Analytics, Tucson, Arizona).

Melting points were determined in a Mel-Temp device from Laboratory Devices (Holliston, Massachusets).

Preparative separations were carried out using a Flash 40 and KP-Sil chromatographic system, 60A (Biotage, Charlottesville, Virginia), a device Chromatotron radial chromatographic (Harrison Research, Palo Alto, California) or by HPLC using a reverse phase column C-18 Typical solvents used were dichloromethane, methanol, ethyl acetate and triethylamine.

Example 2 Synthesis of methyl 1 - [( tert -butyl) oxycarbonyl] -4-oxopiperidin-3-carboxylate

HPLC were carried out in a module Waters 2690 separation, using a ReliaSil column 5-mu C-18 from Column Engineering (50 x 4.6 mm) with a gradient that rises from 5% to 80% acetonitrile in water in 18 minutes

Di- tert-butyl dicarbonate (6.76 g, 3.10 mmol) was added portionwise to a solution of methyl 4-oxopiperidine-3-carboxylate (5.0 g, 25.8 mmol), sodium hydroxide (1.24 g, 31.0 mmol) in 1,4-dioxane and water (60 ml: 30 ml). The resulting solution was stirred at room temperature overnight. The reaction mixture was partitioned between ethyl acetate and water. The organic phase was separated and washed with brine, dried (MgSO 4), filtered and concentrated in vacuo to give quantitative yield of 1 - [( tert- butyl) oxycarbonyl] -4-oxopiperidine-3-carboxylate of methyl as a viscous yellow oil. The crude product gave satisfactory NMR and was used in the next step without purification.
fication

Example 3 Synthesis of tert - butyl 2-methylthio-4-oxo-3,5,6,7,8-pentahydropyridine [4,3-d] pyrimidin-6-carboxylate

The suspension containing methyl 1 - [( tert- butyl) oxycarbonyl] -4-oxopiperidine-3-carboxylate (6.64 g, 25.8 mmol), 2-methyl sulfate was heated at 80 ° C overnight 2-thiopseudourea (7.19 g, 25.8 mmol) and cesium carbonate (16.8 g, 51.6 mmol) in 1-methyl-2-pyrrolidinone (100 ml). Water was added to the reaction mixture to give a homogeneous solution, which was adjusted to pH 4 or 5 using glacial acetic acid. White precipitate formed during this process. The mixture was filtered. The collected solid was washed with water and ethyl acetate to give tert - butyl 2-methylthio-4-oxo-3,5,6,7,8-pentahydropyridine [4,3-d] pyrimidin-6-carboxylate (3 , 0 g) as a white powder. The filtrate was partitioned between ethyl acetate and water. The organic phase was separated and washed with brine, dried (MgSO4) and filtered. Concentration of the product filtered under vacuum gave a yellow solid, which was triturated with ether to give more 2-methylthio-4-oxo-3,5,6,7,8-pentahydropyridine [4,3-d] pyrimidin-6- tert -butyl carboxylate (1.0 g, total yield 52%) as a white fluffy powder.

HPLC [AZ-S procedure], 6.78 min (100%)

MS (M + H / Z), 298.

Example 4 Synthesis of tert - butyl 4-chloro-2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-6-carboxylate

To a solution of tert - butyl 2-methylthio-4-oxo-3,5,6,7,8-pentahydropyridine [4,3-d] pyrimidin-6-carboxylate (820 mg, was added dropwise) 2.76 mmol) in dichloromethane (50 ml) oxalyl chloride (261 ml, 3.03 mmol). To this, two drops of N, N-dimethylformamide were added and the resulting solution was slowly heated to room temperature for 1 hour. The concentration of the reaction mixture gave a solid / yellow oil mixture, which was filtered through a bed of silica gel and washed with ether. Concentration of the filtrate and washings gave tech-butyl 4-chloro-2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-6-carboxylate (850 mg, 97 yield %) as a yellow sticky foam.

HPLC [AZ-S procedure], 12.04 min (100%)

MS (M + H / Z), 316.

Example 5 Synthesis of 4- (2,4-dichlorophenyl) -2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate

A mixture of tec-butyl 4-chloro-2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-6-carboxylate (334 mg, 1) was refluxed under nitrogen , 06 mmol), 2,4-dichlorobenzeneboronic acid (222 mg, 1.16 mmol), bis (dibenzylidenacetone) palladium (0) (48 mg, 0.053 mmol) and 1,4-bis (diphenylphosphino) butane (45 mg, 0.11 mmol) in toluene (2 ml), aqueous 1M sodium carbonate solution (1 ml) and ethanol (0.5 ml). The reaction mixture was then cooled, water was added, and the toluene phase was separated. The aqueous phase was extracted with ethyl acetate and the combined organic extracts were dried (MgSO4), filtered, and the filtrate was concentrated in vacuo. The crude product was purified by column chromatography using 15% acetone in hexanes as eluent, giving 4- (2,4-dichlorophenyl) -2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] tert -butyl pyrimidin-6-carboxylate (471 mg, yield> 100%) as a slightly yellow sticky foam.

HPLC [AZ-S procedure], 14.09 min (75%); MS (M + H / Z), 426.

Example 6 Synthesis of 4- (2,4-dichlorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate

Was added to a solution of 4- (2,4-dichlorophenyl) -2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate (383 mg, 0.898 mmol) in dichloromethane (10 ml) 3-chloroperbenzoic acid (680 mg, 57-86%, 2.25 mmol) in portions. The mixture was stirred at room temperature for 4 hours, poured into a separatory funnel containing 10% aqueous sodium carbonate solution and dichloromethane. The organic phase is separated, washed with 10% sodium sulphite, brine, dried (MgSO4) and filtered. Concentration of the filtrate gave 4- (2,4-dichlorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-6-carboxylic acid tert - butyl ester (430 mg , gross yield> 100%) as a slightly yellow foam / solid.

HPLC [AZ-S procedure], 11.93 min (83%);

MS (M + H / Z), 458.

Example 7 General procedure for displacement of the amine from the sulfone, elimination of the Boc group and salt preparation hydrochloric acid of the final product

A solution was heated at 80 ° C overnight of sulfone (1 mmol) and amine (2 mmol) in acetonitrile (1 ml). The concentration of the reaction mixture gave crude product, which purified by flash chromatography using 3% methanol in  dichloromethane as eluent. The resulting product thus obtained is then dissolved in acetonitrile (4 ml) and aqueous hydrochloric acid (0.5 ml, 37%). The homogeneous solution was stirred at temperature overnight and lyophilized to provide the final product as a salt of hydrochloric acid.

The following compounds were prepared using the previous procedure:

Hydrochloride [4- (2,4-Dichlorophenyl) (5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-2-yl)] {2 - [(5-nitro (2-pyridyl) amino] ethyl }amine

The reaction of 4- (2,4-dichlorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate and 2- (2- aminoethylamino) -5-nitropyridine gave 4- (2,4-dichlorophenyl) -2 - ({2 - [(5-nitro (2-pyridyl)) - amino] ethyl} ami-
no) -5,6,7,8-tetrahydropyridine [4,3-d] tert - butyl pyrimidin-6-carboxylate.

HPLC [AZ-S procedure], 11.83 min (91%);

MS (M + H / Z), 560.

Treatment with HCl and lyophilization gave hydrochloride [4- (2,4-Dichlorophenyl) (5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-2-yl)] {2 - [(5-nitro (2-pyridyl) amino] ethyl }amine.

HPLC [AZ-S procedure], 6.52 min (80%);

MS (M + H / Z), 460.

Hydrochloride {2 - [(6-amino-5-nitro (2-pyridyl)) amino] ethyl} [4- (2,4-dichlorophenyl) (5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin -2-il)] amine

The reaction of 4- (2,4-dichlorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate and 2-amino-3 -nitro-6- (2-amino-ethylamino) pyridine gave 2 - ({2 - [(6-amino-5-nitro (2-pyridyl)) amino] ethyl} amino) -4- (2,4-dichlorophenyl ) -5,6,7,8-tetrahydropyridine [4,3-d] tert - butyl pyrimidin-6-carboxylate.

HPLC [AZ-S procedure], 10.98 min (100%);

MS (M + H / Z), 575.

Treatment with HCl and lyophilization gave hydrochloride {2 - [(6-amino-5-nitro (2-pyridyl)) amino] ethyl} [4- (2,4-dichlorophenyl) (5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin -2-yl)] amine.

HPLC [AZ-S procedure], 5.79 min (95%);

MS (M + H / Z), 475.

Hydrochloride 6 - [(2 - {[4- (2,4-dichlorophenyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-2-yl] amino} ethyl) amino] pyridin-3- carbonitrile

The reaction of 4- (2,4-dichlorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate and 2- (2- aminoethylamino) -5-cyanopyridine gave 4- (2,4-dichlorophenyl) -2 - ({2 - [(5-cyano (2-pyridyl)) - amino] ethyl}
amino) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate.

HPLC [AZ-S procedure], 10.66 min (100%);

MS (M + H / Z), 540.

Treatment with HCl and lyophilization gave hydrochloride 6 - [(2 - {[4- (2,4-dichlorophenyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-2-yl] amino} ethyl) amino] pyridin-3- carbonitrile

HPLC [AZ-S procedure], 5.18 min (80%);

MS (M + H / Z), 440.

Example 8 Synthesis of 4- (2,4-dichlorophenoxy) -2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate

A mixture of tert - butyl 4-chloro-2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-6-carboxylate (100 mg, 0.317 mmol) was heated at reflux overnight , 2,4-dichlorophenol (57 mg, 0.348 mmol) and sodium bis (trimethylsilyl) amide (1.0 M solution in THF, 412 µL, 0.412 mmol) in tetrahydrofuran (2 ml). The mixture was partitioned between ethyl acetate and water. The organic phase was separated and washed with brine, dried (MgSO4) and filtered. Concentration of the filtrate gave crude product (180 mg), which was purified by flash chromatography (0.5% methanol in dichloromethane as eluent) to give 4- (2,4-dichlorophenoxy) -2-methylthio-5,6 , Tert- butyl 7,8-tetrahydropyridine [4,3-d] pyrimidin-6-carboxylate as a white foam (52 mg, 37% yield).

HPLC [AZ-S procedure], 15.93 min (100%);

MS (M + H / Z), 442.

Example 9 Synthesis of 4- (2,4-dichlorophenoxy) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate

Following Example 6, oxidation of 4- (2,4-dichlorophenoxy) -2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate (52 mg , 0.118 mmol) with 3-chloroperbenzoic acid gave 4- (2,4-dichlorophenoxy) -
2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate in quantitative yield
tivo.

HPLC [AZ-S procedure], 12.85 min (95%);

MS (M + H / Z), 474.

Example 10 Synthesis of hydrochloride {2 - [(6-amino-5-nitro (2-pyridyl)) amino] ethyl} {4 - [(2,4-dichlorophenyl) -oxy] (5,6,7,8-tetrahydropyridine [4.3 -d] pyrimidin-2-yl)} amine

Following Example 7, treatment of 4- (2,4-dichlorophenoxy) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate with 2-amino-3-nitro-6- (2-aminoethylamino) pyridine gave 2 - ({2 - [(6-amino-5-nitro (2-iridyl)) amino] ethyl} amino) -4- (2, 4-dichlorophenoxy) -5,6,7,8-tetrahydropyrido [4,3-d] pyrimidine-6-carboxylate.

HPLC [AZ-S procedure], 10.76 min (95%);

MS (M + H / Z), 591.

The treatment with HCl and lyophilization gave the hydrochloride {2 - [(6-amino-5-nitro (2-pyridyl)) amino] ethyl} {4 - [(2,4-dichlorophenyl) oxy] (5,6,7,8-tetrahydropyridine [4,3- d] pyrimidin-2-yl)} amine

HPLC [AZ-S procedure], 5.18 min (99%);

MS (M + H / Z), 491.

Example 11 Synthesis of tert - butyl 4 - [(2,4-dichlorophenyl) amino] -2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-6-carboxylate

A mixture of tert - butyl 4-chloro-2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-6-carboxylate (100 mg, 0.317 mmol) was heated at reflux overnight , 2,4-dichloroaniline (56 mg, 0.348 mmol) and sodium bis (trimethylsilyl) amide (1.0 M solution in THF, 412 µL, 0.412 mmol) in tetrahydrofuran (2 ml). The mixture was partitioned between ethyl acetate and water. The organic phase was separated and washed with brine, dried (MgSO4) and filtered. Concentration of the filtrate gave the crude product, which was purified by flash chromatography (0.5% methanol in dichloromethane as eluent) to give 4 - [(2,4-dichlorophenyl) amino] -2-methylthio-5,6 , Tert- butyl 7,8-tetrahydropyridine [4,3-d] pyrimidin-6-carboxylate as a light yellow oil (85 mg, 61% yield).

HPLC [AZ-S procedure], 9.88 min (100%);

MS (M + H / Z), 441.

Example 12 Synthesis of tert - butyl 4 - [(2,4-dichlorophenyl) amino] -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-6-carboxylate

Following example 6, the oxidation of tert - butyl 4 - [(2,4-dichlorophenyl) amino] -2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-6-carboxylate (84.7 mg, 0.192 mmol) with 3-chloroperbenzoic acid gave 4 - [(2,4-dichlorophenyl) amino] -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] tert -butyl pyrimidin-6-carboxylate in quantitative yield.

HPLC [AZ-S procedure], 11.40 min (95%);

MS (M + H / Z), 473.

Example 13 Synthesis of hydrochloride {2 - [(6-amino-5-nitro (2-pyridyl)) amino] ethyl} {4 - [(2,4-dichlorophenyl) amino] (5,6,7,8-tetrahydropyridine [4,3- d] pyrimidin-2-yl)] amine

Following example 7, the treatment of tert 4 - [(2,4-dichlorophenyl) amino] -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-6-carboxylate -butyl with 2-amino-3-nitro-6- (2-aminoethylamino) pyridine gave 2 - ({2 - [(6-amino-5-nitro (2-pyridyl)) amino] ethyl} amino) -4- [(2,4-dichlorophenyl) amino] (5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate.

HPLC [AZ-S procedure], 9.47 min (95%); MS (M + H / Z), 590.

Treatment with HCl and lyophilization gave hydrochloride {2 - [(6-amino-5-nitro (2-pyridyl)) amino] ethyl} {4 - [(2,4-dichlorophenyl) amino] (5,6,7,8-tetrahydropyridine [4,3- d] pyrimidin-2-yl)] amine.

HPLC [AZ-S procedure], 4.81 min (99%);

MS (M + H / Z), 490.

Example 14 Synthesis of 4- (2,4-difluorophenyl) -2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate

Following example 5, the coupling of tert - butyl 4-chloro-2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-6-carboxylate (1.94 mg, 6.14 mmol) with 2,4-dichlorobenzeneboronic acid (1.07 g, 6.76 mmol) gave 4 - [(2,4-difluorophenyl) -2-methylthio-5,6,7,8-tetrahydropyridine [4,3- d] tert - butyl pyrimidin-6-carboxylate as a white foam (1.54 g, 64% yield).

HPLC [AZ-S procedure], 13.21 min (95%);

MS (M + H / Z), 394.

Example 15 Synthesis of 4- (2,4-difluorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate

Following Example 6, oxidation of 4- (2,4-difluorophenyl) -2-methylthio-5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate (1, 54 g, 3.91 mmol) with 3-chloroperbenzoic acid (2.48 g, 8.61 mmol) gave 4- (2,4-difluorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] tert - butyl pyrimidin-6-carboxylate as a white foam in quantitative yield.

HPLC [AZ-S procedure], 10.34 (95%);

MS (M + H / Z), 426.

Example 16 Synthesis of 4- (2,4-Dichlorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine

He was added to a solution of 4- (2,4-dichlorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine-6-carboxylate (100 mg , 0.218 mmol) in dichloromethane (2 ml) trifluoroacetic acid (1 ml) with care. The resulting solution was stirred at room temperature for 2 hours, concentrated and dried under high vacuum to give 4- (2,4-dichlorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4, 3-d] pyrimidine in quantitative.

HPLC [AZ-S procedure], 4.74 (86%);

MS (M + H / Z), 358.

Example 17 Synthesis of 6-acetyl-4- (2,4-dichlorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine

It was added dropwise to a solution of 4- (2,4-Dichlorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine (100 mg, 0.299 mmol), triethylamine (78 µL, 0.588 mmol) in dichloromethane (2 ml) acetic anhydride (53 µL, 0.588 mmol). Be The resulting mixture was stirred at room temperature overnight. The mixture was partitioned between ethyl acetate and water. The organic phase and washed with brine, dried (MgSO4) and dried filter. The concentration of the filtrate gave the product. stupid 6-acetyl-4- (2,4-dichlorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine in quantitative performance.

HPLC [AZ-S procedure], 7.09 (81%);

MS (M + H / Z), 400.

Example 18 Synthesis of 6-acetyl-2 - ({2- [6-amino-5-nitro (2-pyridyl)) amino] ethyl} amino) -4- (2,4-dichlorophenyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine

Following example 7, the reaction of 6-acetyl-4- (2,4-dichlorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine and with 2-amino -3-nitro-6- (2-aminoethylamino) pyridine gave 6-acetyl-2 - ({2- [6-amino-5-nitro (2-pyridyl)))
amino] ethyl} amino) -4- (2,4-dichlorophenyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine.

HPLC [AZ-S procedure], 7.23 (96%);

MS (M + H / Z), 517.

Example 19 Synthesis of 6 - [(2 - {[6-acetyl-4- (2,4-dichlorophenyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-2-yl] amino} ethyl) amino] pyridin-3-carbonitrile

Following example 7, the reaction of 6-acetyl-4- (2,4-dichlorophenyl) -2- (methylsulfonyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidine Y 2- (2-aminoethylamino) -5-cyanopyridine it gave 6 - [(2 - {[6-acetyl-4- (2,4-dichlorophenyl) -5,6,7,8-tetrahydropyridine [4,3-d] pyrimidin-2-yl] amino} ethyl) amino] pyridin-3-carbonitrile.

HPLC [AZ-S procedure], 6.87 (90%);

MS (M + H / Z), 482.

Example 20 Monitoring of GSK3 inhibitory activity using an assay without cells

The pyrimidine and pyridine compounds of the present invention were dissolved in DMSO, then tested for inhibition of human GSK3β (the nucleotide sequence for human GSK3β appears in GenBank with accession number L33801). The expression of GSK3β is described, for example, in Hughes et al., Eur J. Biochem. , 203 : 305-11 (1992), which is incorporated herein by reference.

A 300 µl aliquot of buffer was dispensed substrate (30 mM tris-HCl, 10 mM MgCl 2, DTT 2 mM, 3 µg / ml GSK3β and CREB peptide bound to SGSG 0.5 µM biotinylated prephosphorylated (Chiron Technologies PTY Ltd., Clayton, Australia) to wells of a microtiter plate 96 well polypropylene. 3.5 µl / well of DMSO was added which contains varying concentrations of each compound that has been to test or staurosporine (a known kinase inhibitor used as a positive control, or a negative control (i.e., DMSO only), and it mixed completely. Then the reactions started by adding 50 µl / well of 1 µM unlabeled ATP and 1 to 2 x 10 7 cpm of ATP labeled with γ33 P, and let the reaction progress for about three hours at room temperature.

While the reaction took place they were blocked "Combiplate 8" capture plates of coated Labsystems of streptavidin (Labsystems, Helsinki, Finland) by incubation of these with 300 µl / well of PBS containing 1% bovine serum albumin for at least one hour at room temperature. The blocking solution was then removed by aspiration, and the capture plates were filled with 100 µl / well of reagent reagent (ATP 50 / EDTA 20 mM).

When the enzymatic reaction of three hours, aliquots of 100 µl were transferred in triplicate of each reaction mixture to three wells containing solution of detention, a well in each of the three capture plates, and the well contents were mixed well. After an hour at room temperature the wells of the plates were emptied capture by aspiration and washing five times using PBS and a 12 channel Corning 430474 ELISA plate washer. Finally I know added 200 µl scintillation fluid Microscint-20 to each well of the plate. Be they coated the plates with plate sealants, then left in a stirrer for 30 minutes. Each plate was counted capture on a Packard TopCount scintillation counter (Meridian, Connecticut) and the results were represented as a function of The concentration of compound.

The compounds of the present invention regarding the inhibitory activity against GSK3 according to this test. The compounds of examples 3 a 19 showed IC 50 of 1 µM or less relative to GSK3 in This test without cells.

In accordance with the above, these results demonstrate that the compounds of the present invention show inhibitory activity against GSK3.

Example 21 Classification of GSK3 inhibitory activity using a cell-based glycogen synthase assay

CHO-HIRC cells are maintained in 10 cm tissue culture plates in Ham / serum F12 medium 10% dialyzed fetal bovine. Plaque cells are harvested 10 cm confluent and divided into the 6 wells of a plate 6-well tissue culture to a final volume of 2 ml of means, medium. The cells are allowed to grow at 37 ° C for 24 hours. Be then wash the cells three times in Ham's F12 medium that doesn't it contains fetal bovine serum and finally the cells are left for an additional 24 hours at 37 ° C in 2 ml of the medium without serum.

At the end of this time 20 µl of compound dissolved in DMSO to each well and incubated at 37 ° C. After 20 minutes the medium is removed and the cells are washed once in PBS at room temperature and then freeze quickly on plates in liquid nitrogen. Defrost then the cells on ice in the presence of 140 µl of buffer of lysis (50 mM tris pH 7; 1 mM EDTA, 100 mM NaF, leupeptin 25 µg / ml, 1 mM DTT, 1 mM PMSF) per well. The cells are scraped of the plates and freeze in Eppendorf tubes on dry ice. Be then defrost the lysates and thaw on dry ice.

After re-defrosting they stir the lysates at 14,000 g for 15 minutes. They are then taken supernatants and stored on ice. Each supernatant (45 µl) is added to 45 µl of reaction buffer (65 mM Tris pH 7.8; 26 mM EDTA, 32.5 mM KF, 9.3 mM UDP-glucose; glycogen 11 mg / ml; 14 C-UDP-glucose 500 nCi / ml) and 45 µl more is added to 45 µl of buffer 20 mM reaction / glucose-6-phosphate. Be incubate the reactions at 30 ° C for 30 minutes and then splash on a 2 cm square 31ET chromatographic paper (Whatman). Be wash the filter papers twice for 20 minutes in ethanol 66%, washed briefly in acetone and dried for 1 hour at room temperature.

The filters are added to 5 ml of material liquid scintillation and counted in a scintillation counter. Be expresses the percentage of total glycogen synthase that is active in any lysate as 100X (cpm less glucose-6-phosphate) / (cpm more glucose-6-phosphate). Such values are determined in duplicate for 5 different concentrations of compound and for DMSO only, and then the values are represented against the logarithm of concentration. The concentration of compound that stimulates glycogen synthase activity until 50% of the maximum level is determined by adjusting a curve Sigmoidal to the data represented. The maximum level is defined as that level at which glycogen synthase activity tends asymmetrically when the concentration of the test compound increases substantially beyond the EC50.

Example 22 Follow-up of protein phosphorylation inhibition tau A. Transient transfection of COS cells with plasmid GSK3 expression and tau expression Plasmid Construction

COS cells are maintained in culture flasks of T25 tissue in MEM medium with a large amount of glucose / bovine serum 5% fetal. Cells from a confluent T25 flask are harvested and plant 80,000 cells / well in 6 tissue culture plates Corning wells in a final volume of 2 ml / well of medium. Be let the cells grow at 37 ° C for 48 hours. They wash later the cells twice in Opti-MEM that does not contain fetal bovine serum, and finally the cells are left in 1 ml of Opti-MEM.

The polynucleotide encoding the tau protein in plasmid pSG5 is subcloned under an early SV40 promoter to generate a tau expression plasmid. Cloning of the cDNA encoding the tau protein is generally described by Goedert et al., EMBO Journal , 8 (2): 393-399 (1989), which is incorporated herein by reference. A GSK3 expression plasmid is prepared by polynucleotide subcloning encoding GSK3β in pCG, which is an ApEVRF derivative described by Giese et al., Genes & Development , 9 : 995-1008 (1995) and Matthias et al. , Nucleic Acid Research , 17 : 6418 (1989), both are incorporated herein by reference.

The following solutions are prepared in tubes 1.5 ml Eppendorf: solution A: for each transfection, they are diluted 2 µg of DNA (tau expression plasmid) and 0.7 µg of DNA (GSK3 expression plasmid) in 100 µl of Opti-MEM (Gibco BRL); solution B: for each transfection, 8 µl of lipofectamine reagent is diluted in 100 µl of Opti-MEM. They combine the two solutions, mix vigorously, and incubate at temperature room for 45 minutes to allow complexes to form of DNA-liposome. 0.8 is added for each transfection ml of Opti-MEM to the tube containing the complexes. The diluted solution is vigorously mixed and spread over the washed cells. The cells are incubated with the DNA / lipofectamine complexed for 6 hours at 37 ° C in a CO2 incubator. After incubation is added 1 ml of growth medium (MEM with high glucose content) with 20% FBS to each well and incubate at 37 ° C overnight. The medium is replaced with fresh medium complete at 18 hours after the start of transfection, and let the cells grow at 37 ° C for another 48 hours.

B. Tau phosphorylation inhibition assay

Two hours before harvest, 2 \ mul are added of test compound (GSK3 inhibitor) dissolved in DMSO at each well and incubate at 37 ° C. After 2 hours the medium is removed and quickly freeze the cells in the plates on ice dry and stored at -70 ° C. Defrost cells on ice in the presence of 200 µl of lysis buffer (Triton® 1% X-100, 20 mM Tris pH 7.5, 137 mM NaCl, 15% glycerol, 25 µg / ml leupeptin, 1 µml ml of pepstatin-A, 1 µM PMSF, 21 µg / ml of aproptinin, 50 mM NaF, β-glycerophosphate 50 mM, 15 mM sodium pyrophosphate, 1 mM sodium orthovanadate). Be centrifuge the contents of each well at 14,000 g, at 4 ° C for 5 minutes and the supernatants are transferred to tubes clean. At this time the lysates can be stored at -20 ° C.

C. ELISA to detect phosphorylated tau in lysates of cells

4 Immulon (Dynatech) strips are coated with tau monoclonal antiphosphorylated (AT8, Polymedco, Inc.) at 5 µg / ml in PBS containing Ca ++ and Mg ++, 100 µg / well. After overnight incubation at 4 ° C, the strips are washed twice with wash buffer (PBS containing 0.05% Tween® 20) and crashes with PBS containing 1% BSA, 5% normal mouse serum and 0.05% Tween® 20 at room temperature for 1 hour. Washing Strips 5 times with wash buffer. Lysate is added (100 µl) diluted to 1:10 in PBS containing 1% BSA, NaN3 at 0.1% to each well and incubate at room temperature for one hour. After washing, 100 µl of tau is added to each well biotinylated monoclonal anti (non-phosphorylated) 0.5 µg / ml (HT7, Polymedco, Inc.) in PBS-BSA. Be wash the strips 5 times and add conjugated streptavidin with HRP, incubated at room temperature for 30 minutes and washed extensive form with wash buffer. TMB substrate (Pierce) is used for color development and the reaction is stopped by addition of an equal volume of 0.8 M sulfuric acid. strips on an ELISA plate reader using a 450 nm filter. Be determines the concentration of the compound that inhibits the phosphorylation of tau at 50% of the maximum level (i.e. IC_ {50}) by adjusting a sigmoidal curve to the data represented.

Example 23 Test of the potential of GSK3 inhibitors to protect the cells of the primary hippocampus of the excitotoxicity of glutamate

Hippocampi were dissected from embryonic rats for 18 to 19 days. The tissue was collected in HibernateTM medium (Gibco BRL) and crumbled into pieces of approximately 1 mm. The tissue is dissociated using the Papain dissociation system (Worthington Biochemical Corporation). After isolation, the cells were resuspended in serum-free medium consisting of Neurobasal ™ (Gibco BRL), 2% B27 supplement (Gibco BRL), L-glutamine and antibiotics. The cells were placed in 35 mm tissue culture discs coated with poly-L-lysine at a concentration of 7.5 x 10 4 cells per disc. After 10 to 14 days at 37 ° C in 5% CO2 the cells were washed and fed with fresh medium. The following day representative compounds of the invention were added to the culture medium to a final concentration of between 1 nM and 100 µM. Four to eight hours after the compound was added, the conditioned medium was removed from the cells and stored at 27 ° C. Cultures were washed twice with balanced HEPES buffered saline (HBSS) containing 10 µM glycine. Grabb and Choi, J.Neuroscience 19 : 1657-62 (1999). The cultures were then exposed for 5 minutes at room temperature to 200 µM glutamic acid in the same HBSS. After exposure, the cultures were washed three times with the buffer and then returned to their original conditioned medium containing the compounds. Twenty to twenty four hours after exposure to glutamic acid, the cultures were washed in HBSS and exposed for 10 minutes to trypan blue. This dye is captured by dead cells. The cultures were washed and then fixed for 30 minutes in 4% paraformaldehyde. The number of live and dead large neurons (blue nuclei) (at least 200 cells of each culture) is counted by phase contrast microscopy and photographed. Using this procedure the compounds of this invention have been shown to be able to significantly reduce the potential of glutamate to induce neuronal cell death.

Example 24 Evaluation of efficacy in diabetic rodents (The glucose tolerance) Formulating compound for dosage orally

Typically, compounds of test for oral use as solutions in water or suspensions in 1% carboxymethylcellulose / tween-80 0.1% (both of Sigma Chem., MO) the day before administration. Some early compounds were formulated as 15% Captisol solutions (a cyclodexitrin modified by CyDex Co., IL) following procedures common to the following. For water solutions, the compound powder is solubilized. dry and lyophilized test in distilled water and mix well by agitation and ultrasound. If necessary, the pH of the test solution with 1 N NaOH or 1 N HCl and filtered finally in sterile conditions through a syringe equipped with a 0.2 cellulose acetate membrane micrometers (Millipore Co., MA). For oral administration, mix the powder of the test compound with a fresh suspension of 1% carboxymethyl cellulose / 0.1% tween-80 and extensively ultrasound, the pH is adjusted in case of need as described above, and stir until the particle size is homogeneous and <10 micrometers of size.

Glucose tolerance test in diabetic mice:

Obese db / db mice (C57BlKs / J were acquired females) to Jackson Labs (Bar Harbor, ME) 8 weeks old and they used for the efficacy test 1 to 2 weeks later. In the morning of a rehearsal the power was withdrawn soon in the morning (7 to 8 hours before the bolus of glucose). Local anesthesia was applied (EMLA cr \ thetame, Astra Pharm, MA) at the tail end and it they took blood samples from 50 to 100 µl of cuts in the tip of the tail and collected in Eppendorf tubes containing 5 µm of 500 U / ml sodium heparin (Elkins-Sinn, NJ) with the subsequent plasma isolation. Samples were taken at different intervals throughout the day for a total of 6 to 8 moments Temporary Mice were randomly grouped into groups of treatment and were given the first oral dose of the test compound (0.2 ml volume) 4.5 hours before the glucose and again 0.5 hours before the administration of 0.2 ml of 50% dextrose (Abbott Lab., IL) by taking by mouth (oGTT) or intraperitoneal injection. After the sample of final blood approximately 2 hours after administration of glucose, the food was returned to the animals.

Regulation of baseline glycemia and insulinemia:

They were typically administered orally test compounds to db / db mice (see above) or to ZDF rats (Genetic Models, Inc .; Indianapolis, IN) in context of a multi-day, multi-dose regimen or as a single bolus. ZDF rats were received at 8 weeks of age and were used for the efficacy test 1 to 2 weeks after. The food was removed approximately 30 minutes before the dosage and a single bolus of the compound of assay (dosage volume ranging from 1 to 8 mg / ml). Is taken blood samples as described previously in 1 to 6 Temporary moments for the next 2 to 3 hours. It was returned feed to animal cages after sampling blood.

Primary endpoints

Glucose and insulin levels of plasma and / or blood samples. Glucose levels are measured. in whole blood using the One-Touch glucometer (Lifescan Co., CA) and in plasma using the glucose analyzer Beckman Glucose results typically reflect values in mouse blood and plasma values for rat studies. The Measurement of insulin levels was by ELISA (Crystal Chem. Co., IL) following the protocol of the supplier.

Quantification of the results

Efficacy can be expressed as mg / dL of glucose or ng / ml insulin or represent as the area under the curve (AUC) for plasma glucose (taken above baseline 100 mg / dL normoglycemic) and insulin (taken above baseline normoinsulinemic of 1 ng / ml). Typically, when expressed as AUC, the results are represented exactly as AUC reduced ([Vehicle control AUC - test group AUC) / AUC of vehicle control x 100]). Such an expression provides a Unique quantitative expression of the greatest elimination of glucose and / or reduced baseline hyperglycemia or insulin conservation compared to placebo control group.

Results

Representative compounds of the invention showed good potency in vitro , and when formulated in captisol and administered subcutaneously to mice (30 mg / kg), they showed high bioavailability and tissue penetration in vivo . A significant reduction in baseline hyperglycemia was observed just before the glucose tolerance test, and significantly improved glucose elimination after glucose exposure. A 45-50% reduction in AUC was observed with respect to the control group if the glucose response is quantified by determining the area under the blood glucose curve (AUC) from -60 minutes to +120 minutes. This is comparable to the efficacy obtained with troglitazone (when dosed orally for at least several days either at 60 or 100 mg / kg / day). Also significant was the observation that insulin levels in treated animals remained below those of control mice.

Claims (63)

1. A compound of structure (I):

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25

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in the that: X and Y are nitrogen; A 1 is aryl or heteroaryl optionally replaced; A2 is aryl, aryloxy, arylamino or optionally substituted heteroaryl; R1, R2, R3 and R4 are independently selected from the group consisting of hydrogen, hydroxyl, and lower alkyl, lower cycloalkyl, alkylaminoalkyl, lower alkoxy, amino, alkylamino, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, aryl and heteroaryl optionally substituted; R '1, R' 2, R '3 and R' 4 are independently selected from the group consisting of hydrogen, and optionally substituted lower alkyl; R 5, R 6 and R 7 are selected regardless of the group consisting of hydrogen, hydroxy, halo, carboxyl, nitro, amino, amido, amidino, imido, cyano and lower alkyl substituted or unsubstituted, lower alkoxy, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, alkylaminocarbonyloxy, arylaminocarbonyloxy, formyl, alkyl lower carbonyl, alkoxy lower-carbonyl, aminocarbonyl, aminoaryl, alkylsulfonyl, sulfonamido, aminoalkoxy, alkylamino, arylamino, aralkylamino, heteroarylamino, heteroaralkylamino, alkylcarbonylamino, alkylaminocarbonylamino, arylaminocarbonylamino, aralkylcarbonylamino, heteroaralkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, amidino, cycloalkyl, cycloamido, cyclothioamido, cycloamidino, heterocycloamidino, cycloimido, heterocycle, guanidinyl, aryl, biaryl, heteroaryl, heterobaryl, heterocycle, heterocycloalkyl, arylsulfonyl and arylsulfonamido; and the pharmaceutical salts thereof. 2. A compound of claim 1, wherein at least one of A 1 and A 2 is an aromatic ring having 3 to 10 carbon atoms in the ring and optionally 1 or more ring heteroatoms. 3. A compound of claim 1, wherein A2 is optionally aryl, arylamino or carbocyclic aryloxy replaced. 4. A compound of claim 2, wherein at least one of A 1 and A 2 is optionally heteroaryl replaced. 5. A compound of claim 2, wherein A2 is selected from the group consisting of phenylamino and substituted or unsubstituted phenyloxy. 6. A compound of claim 2, wherein at least one of A_ {1} and A_ {2} is selected from the group constituted by pyridyl, pyrimidinyl, thiazolyl, indolyl, imidazolyl, oxadiazolyl, tetrazolyl, pyrazinyl, triazolyl, thiophenyl, furanyl, quinolinyl, purinyl, naphthyl, benzothiazolyl, benzopyridyl and benzimidazolyl substituted or not replaced. 7. A compound of claim 2, wherein at least one of A_ {1} and A_ {2} is substituted with at least one and no more than 3 substitution groups. 8. A compound of claim 7, wherein said substitution groups are independently selected from group consisting of nitro, amino, cyano, halo, thioamido, amidino, oxamidino, alkoxyamidino, imidino, guanidino, sulfonamido, carboxyl, formyl, lower alkyl, lower haloalkyl, alkoxy lower, lower haloalkoxy, lower alkoxyalkyl, alkyl lower-lower aminoalkoxy, alkyl lower carbonyl, aralkyl lower-carbonyl, heteroaralkyl lower carbonyl, alkylthio, aminoalkyl and cyanoalkyl. 9. A compound of claim 2, wherein A_ {2} has the formula:

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in which L is -NH- or -O-, Y R_ {8} and R_ {9} are selected regardless of the group consisting of hydrogen, nitro, amino, cyano, halo, thioamido, amidino, oxamidino, alkoxyamidino, imidino, guanidinyl, sulfonamido, carboxyl, formyl, alkyl lower, lower haloalkyl, lower alkoxy, haloalkoxy lower, lower alkoxyalkyl, alkyl lower-lower aminoalkoxy, alkyl lower carbonyl, aralkyl lower-carbonyl, heteroaralkyl lower carbonyl, alkylthio, aryl and aralkyl 10. A compound of claim 9, in the that R_ {8} and R_ {9} are selected from the group consisting of halo and lower haloalkyl. 11. A compound of claim 10, in the that R 8 and R 9 are halo. 12. A compound of claim 9, in the that A_ {2} is selected from the group consisting of 2,5-dichlorophenylamino and 2,5-dichlorophenyloxy. 13. A compound of claim 1, in the which at least one of R 1, R 2, R 3 and R 4 is alkyl bottom substituted selected from the group consisting of lower haloalkyl, heterocycloaminoalkyl and alkyl lower-lower aminoalkyl. 14. A compound of claim 13, in the that at least one of R 1, R 2, R 3 and R 4 is alkyl lower-lower aminoalkyl. 15. A compound of claim 13, in the that R 1, R 2 and R 3 are hydrogen and R 4 is selects from the group consisting of hydrogen, methyl, ethyl, aminoethyl, dimethylaminoethyl, pyridylethyl, piperidinyl, pyrrolidinylethyl, piperazinylethyl and morpholinyl ethyl. 16. A compound of claim 1, in the that at least one of R 5 and R 7 is selected from the group constituted by substituted aryl, heteroaryl and biaryl and not replaced. 17. A compound of claim 16, in the that at least one of R 5 and R 7 is a substituted or non-substituted moiety substituted formula: 27 in which R_ {10}, R_ {11}, R 12, R 13 and R 14 are independently selected between the group consisting of hydrogen, nitro, amino, cyano, halo, thioamido, carboxyl, hydroxy and lower alkyl optionally substituted, lower alkoxy, lower alkoxyalkyl, haloalkyl lower, haloalkoxy lower, aminoalkyl, alkylamino, alkylthio, alkylcarbonylamino, aralkylcarbonylamino, heteroaralkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aminocarbonyl, alkyl lower-aminocarbonyl, aminoaralkyl, alkyl lower-aminoalkyl, aryl, heteroaryl, cycloheteroalkyl, aralkyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, arylcarbonyloxyalkyl, alkylcarbonyloxyalkyl, heteroarylcarbonyloxyalkyl, aralkylcarbonyloxyalkyl and heteroaralkylcarbonyloxyalkyl. 18. A compound of claim 17, in the that R 10, R 11, R 13 and R 14 are hydrogen and R 12 is selected from the group consisting of halo, alkyl lower, hydroxy, lower alkoxy, lower haloalkyl, aminocarbonyl, alkylaminocarbonyl and cyano. 19. A compound of claim 17, in the that R 11, R 13 and R 14 are hydrogen and R 10 and R 12 are independently selected from the group consisting of halo, lower alkyl, hydroxy, lower alkoxy, haloalkyl lower and cyano. 20. A compound of claim 17, in the that R 10, R 11, R 13 and R 14 are hydrogen and R 12 is heteroaryl. 21. A compound of claim 17, in the that R 10, R 11, R 13 and R 14 are hydrogen and R 12 is heterocycloalkyl. 22. A compound of claim 17, in the that at least one of R 10, R 11, R 12, R 13 and R 14 are halo and the rest of R 10, R 11, R 12, R 13 and R 14 are hydrogen. 23. A compound of claim 17, in the that at least one of R 5 and R 7 is selected from the group constituted by dichlorophenyl, difluorophenyl, trifluoromethylphenyl, chlorofluorophenyl, bromochlorophenyl, ethylphenyl, methylchlorophenyl, imidazolylphenyl, cyanophenyl, morpholinophenyl and cyanochlorophenyl. 24. A compound of claim 1, in the that R 6 is substituted alkyl selected from the group constituted by aralkyl, hydroxyalkyl, aminoalkyl, aminoaralkyl, carbonylaminoalkyl, alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl, aralkylcarbonylaminoalkyl, aminoalkoxyalkyl and arylaminoalkyl. 25. A compound of claim 1, in the that R 6 is substituted amino selected from the constituted group by alkylamino, alkylcarbonylamino, alkoxycarbonylamino, arylalkylamino, arylcarbonylamino, alkylthiocarbonylamino, arylsulfonylamino, heteroarylamino, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, and heteroaralkylcarbonylamino. 26. A compound of claim 1, in the that R 6 is selected from the group consisting of aminocarbonyl, alkyloxycarbonyl, aryloxycarbonyl, aralkyloxycarbonyl and unsubstituted or substituted alkylaminoalkyloxycarbonyl. 27. A compound of claim 1, in the that R 6 is selected from the group consisting of amidino, guanidino, cycloimido, heterocycloimido, cycloamido, heterocycloamido, cyclothioamido and lower heterocycloalkyl. 28. A compound of claim 1, in the that R 6 is aryl. 29. A compound of claim 1, in the that R 6 is heteroaryl. 30. A compound of claim 29, in the that R 6 is selected from the group consisting of pyridyl, pyrimidinyl, thiazolyl, indolyl, imidazolyl, oxadiazolyl, tetrazolyl, pyrazinyl, triazolyl, thienyl, furanyl, quinolinyl, pyrrolylpyridyl, benzothiazolyl, benzopyridyl, benzotriazolyl and substituted or unsubstituted benzimidazolyl. 31. A compound of structure (I): 28 in the that: X and Y are nitrogen; A 1 is aryl or heteroaryl optionally replaced; A2 is aryl, aryloxy, acylamino or optionally substituted heteroaryl; R1, R2, R3 and R4 are independently selected from the group consisting of hydrogen, hydroxyl, and lower alkyl, lower cycloalkyl, alkylaminoalkyl, lower alkoxy, amino, alkylamino, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, aryl and heteroaryl optionally substituted; R '1, R' 2, R '3 and R' 4 are independently selected from the group consisting of hydrogen, and optionally substituted lower alkyl; R_ {5} and R_ {7} are selected regardless of the group consisting of hydrogen, hydroxy, halo, carboxyl, nitro, amino, amido, amidino, imido, cyano and lower alkyl substituted or unsubstituted, lower alkoxy, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, alkylaminocarbonyloxy, arylaminocarbonyloxy, formyl, alkyl lower carbonyl, alkoxy lower-carbonyl, aminocarbonyl, aminoaryl, alkylsulfonyl, sulfonamido, aminoalkoxy, alkylamino, arylamino, aralkylamino, heteroarylamino, heteroaralkylamino, alkylcarbonylamino, alkylaminocarbonylamino, arylaminocarbonylamino, aralkylcarbonylamino, heteroaralkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, amidino, cycloalkyl, cycloamido, cyclothioamido, cycloamidino, heterocycloamidino, cycloimido, heterocycle, guanidinyl, aryl, biaryl, heteroaryl, heterobaryl, heterocycle, heterocycloalkyl, arylsulfonyl and arylsulfonamido; and pharmaceutically acceptable salts of same. 32. A compound of claim 31, in the that at least one of A 1 and A 2 is an aromatic ring that it has 3 to 10 carbon atoms in the ring and optionally 1 or more heteroatoms in the ring. 33. A compound of claim 32, in the that A 2 is aryl, arylamino or carbocyclic aryloxy optionally substituted. 34. A compound of claim 32, in the that at least one of A 1 and A 2 is optionally heteroaryl replaced. 35. A compound of claim 32, in the that A2 is selected from the group consisting of phenylamino and substituted or unsubstituted phenyloxy. 36. A compound of claim 32, in the that at least one of A_ {1} and A_ {2} is selected from the group constituted by pyridyl, pyrimidinyl, thiazolyl, indolyl, imidazolyl, oxadiazolyl, tetrazolyl, pyrazinyl, triazolyl, thiophenyl, furanyl, quinolinyl, purinyl, naphthyl, benzothiazolyl, benzopyridyl and benzimidazolyl substituted or not replaced. 37. A compound of claim 32, in the that at least one of A_ {1} and A_ {2} is substituted with at least one and no more than 3 substitution groups. 38. A compound of claim 37, in the that said substitution groups are independently selected from the group consisting of nitro, amino, cyano, halo, thioamido, amidino, oxamidino, alkoxyamidino, imidino, guanidino, sulfonamido, carboxyl, formyl, lower alkyl, lower haloalkyl, alkoxy lower, lower haloalkoxy, lower alkoxyalkyl, alkyl lower-aminoalkyl lower-carbonyl, alkylthio, aminoalkyl and cyanoalkyl. 39. A compound of claim 32, in the that at least one of A_ {1} and A_ {2} has the formula: 29 in which L is -NH- or -O-, Y R_ {8} and R_ {9} are selected regardless of the group consisting of hydrogen, nitro, amino, cyano, halo, thioamido, amidino, oxamidino, alkoxyamidino, imidino, guanidinyl, sulfonamido, carboxyl, formyl, alkyl lower, lower haloalkyl, lower alkoxy, haloalkoxy lower, lower alkoxyalkyl, alkyl lower-lower aminoalkoxy, alkyl lower carbonyl, aralkyl lower-carbonyl, heteroaralkyl lower carbonyl, alkylthio, aryl and aralkyl 40. A compound of claim 39, in the that R_ {8} and R_ {9} are selected from the group consisting of halo and lower haloalkyl. 41. A compound of claim 40, in the that R 8 and R 9 are halo. 42. A compound of claim 39, in the that A_ {2} is selected from the group consisting of 2,5-dichlorophenylamino and 2,5-dichlorophenyloxy. 43. A compound of claim 39, in the which at least one of R 1, R 2, R 3 and R 4 is alkyl bottom substituted selected from the group consisting of lower haloalkyl, heterocycloaminoalkyl and alkyl lower-lower aminoalkyl. 44. A compound of claim 43, in the that at least one of R 1, R 2, R 3 and R 4 is alkyl lower-lower aminoalkyl. 45. A compound of claim 43, in the that R 1, R 2 and R 3 are hydrogen and R 4 is selects from the group consisting of hydrogen, methyl, ethyl, aminoethyl, dimethylaminoethyl, pyridylethyl, piperidinyl, pyrrolidinylethyl, piperazinylethyl and morpholinyl ethyl. 46. A compound of claim 31, in the that at least one of R 5 and R 7 is selected from the group constituted by substituted aryl, heteroaryl and biaryl and not replaced. 47. A compound of claim 46, in the that at least one of R 5 and R 7 is a substituted or non-substituted moiety substituted formula: 30 in which R_ {10}, R_ {11}, R 12, R 13 and R 14 are independently selected from the group consisting of hydrogen, nitro, amino, cyano, halo, thioamido, carboxyl, hydroxy and lower alkyl, lower alkoxy, lower alkoxyalkyl, lower haloalkyl, lower haloalkoxy, aminoalkyl, alkylamino, alkylthio, alkylcarbonylamino, aralkylcarbonylamino, heteroaralkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aminocarbonyl, alkyl lower-aminocarbonyl, aminoaralkyl, alkyl lower-aminoalkyl, aryl, heteroaryl, cycloheteroalkyl, aralkyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, arylcarbonyloxyalkyl, alkylcarbonyloxyalkyl, heteroarylcarbonyloxyalkyl, aralkylcarbonyloxyalkyl and heteroaralkylcarbonyloxyalkyl. 48. A compound of claim 47, in the that R 10, R 11, R 13 and R 14 are hydrogen and R 12 is selected from the group consisting of halo, alkyl lower, hydroxy, lower alkoxy, lower haloalkyl, aminocarbonyl, alkylaminocarbonyl and cyano. 49. A compound of claim 47, in the that R 11, R 13 and R 14 are hydrogen and R 10 and R 12 are independently selected from the group consisting of halo, lower alkyl, hydroxy, lower alkoxy, haloalkyl lower and cyano. 50. A compound of claim 47, in the that R 10, R 11, R 13 and R 14 are hydrogen and R 12 is heteroaryl. 51. A compound of claim 47, in the that R 10, R 11, R 13 and R 14 are hydrogen and R 12 is heterocycloalkyl. 52. A compound of claim 47, in the that at least one of R 10, R 11, R 12, R 13 and R 14 are halo and the remainders of R 10, R 11, R 12, R 13 and R 14 are hydrogen. 53. A compound of claim 47, in the that at least one of R 5 and R 7 is selected from the group constituted by dichlorophenyl, difluorophenyl, trifluoromethylphenyl, chlorofluorophenyl, bromochlorophenyl, ethylphenyl, methylchlorophenyl, imidazolylphenyl, cyanophenyl, morpholinophenyl and cyanochlorophenyl. 54. A compound of claim 31, in the that R 6 is substituted alkyl selected from the group constituted by aralkyl, hydroxyalkyl, aminoalkyl, aminoaralkyl, carbonylaminoalkyl, alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl, aralkylcarbonylaminoalkyl, aminoalkoxyalkyl and arylaminoalkyl. 55. A compound of claim 31, in the that R 6 is substituted amino selected from the constituted group by alkylamino, alkylcarbonylamino, alkoxycarbonylamino, arylalkylamino, arylcarbonylamino, alkylthiocarbonylamino, arylsulfonylamino, heteroarylamino, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylcarbonylamino, and heteroaralkylcarbonylamino. 56. A compound of claim 31, in the that R 6 is selected from the group consisting of aminocarbonyl, alkyloxycarbonyl, aryloxycarbonyl, aralkyloxycarbonyl and unsubstituted or substituted alkylaminoalkyloxycarbonyl. 57. A compound of claim 31, in the that R 6 is selected from the group consisting of amidino, guanidino, cycloimido, heterocycloimido, cycloamido, heterocycloamido, cyclothioamido and lower heterocycloalkyl. 58. A compound of claim 31, in the that R 6 is aryl. 59. A compound of claim 31, in the that R 6 is heteroaryl. 60. A compound of claim 59, in the that R 6 is selected from the group consisting of pyridyl, pyrimidinyl, thiazolyl, indolyl, imidazolyl, oxadiazolyl, tetrazolyl, pyrazinyl, triazolyl, thienyl, furanyl, quinolinyl, pyrrolylpyridyl, benzothiazolyl, benzopyridyl, benzotriazolyl and substituted or unsubstituted benzimidazolyl. 61. A pharmaceutical composition comprising an amount of a compound of claim 1 effective for inhibit the activity of GSK3 in a human or animal subject when administer to it, together with a pharmaceutically vehicle acceptable. 62. A compound of claim 1 or claim 31, for use as a pharmaceutical compound. 63. Use of a compound of claim 1 or claim 31 in the manufacture of a medicament for the treatment of diabetes, Alzheimer's disease and other disorders neurodegenerative, obesity, cardiovascular disease atherosclerotic, arterial hypertension, ovarian syndrome polycystic, syndrome X, ischemia, traumatic brain injury, bipolar disorder or cancer.